ge05-gene+regulation+in+eukaryotes(1)

8/10/2019 GE05-Gene+Regulation+in+Eukaryotes(1)

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Gene Regulation in

Eukaryotes

Dr. Syahril AbdullahMedical Genetics Laboratory

[email protected]


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The Genome

[email protected]

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 Enigmathere is no correlation between complexity of an organism and its genome size !!

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


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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

set of genes

Cellular Differentiation in Higher Eukaryotes


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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 transcribed at different stages of

human development


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[email protected]

Lecture Outline

1. Cellular Differentiation in Higher Eukaryotes2. 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 Control3. Review


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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 splicedor 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 proteindegradation, or its subcellular localization


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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 MethylationC. 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.


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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% of 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


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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


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Chromatin: DNA + Histones

i.

Euchromatin = loosely packed, active genesii. Heterochromatin = condensed region, genes

transcriptionally silent. At centromeres

Transcriptionally inactive

Transcriptionally active


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Transcription FactorsRNA Pol

AcetylationTranscription

DNAMethyltransferase

5-methyl-C

Methyl CpG BindingProteins

HistoneDeacetylase

NO TranscriptionDeacetylation

Transcription factors

Chromatin CompactionTranscriptional Silencing

Association between CpG methylation and

histone acetylations

1. Silencing due to the chromatin compaction.

2. Interfere with the entry of transcription

factors.


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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)


H3K9me

H3K4me


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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.

1. Genomic Level Control : (C) Chromatin Remodeling


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Epigenetic Inheritance?

How histone modifications, nucleosome

positioning & other types of epigenetic

marks might be maintained is still

unclear


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5 UTR 3 UTR


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2. Transcriptional Level Control

TATA BoxUpstream

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

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


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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 -- thisactivates expression of the metallothionein gene.

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

or a regulatory hormone


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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


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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 transcriptof fibronectin gene

FibroblastmRNA

Liver mRNA

Exon

EIIIB

Exon

EIIIA

- exons EIIIA and EIIIB arespliced 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


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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.

Ribonucleasesare 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 CaseinDNA

mRNA Casein

Gene for CaseinDNA

mRNA CaseinRibonuclease

Digest

Milk

Gene for CaseinDNA

mRNA Casein

Ribonuclease

ProlactinPreventsDigestion


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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 3UTR) regulate gene expression inresponse 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)


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4. Translational Level Control

[email protected]

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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Review


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Dr. Syahril AbdullahMedical Genetics Laboratory

[email protected]

The EndLets have a 5 min break before my next class!

ge05-gene+regulation+in+eukaryotes(1)

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