Differential Gene Expression Mechanisms & Development

Differential Gene Expression Mechanisms & Development

Chapter 5

Developmental Genetics

•Investigates how genotypes transformed into phenotype during development

•Examines differential gene expression in different types of cells

Levels at which gene expression can be regulated

•Genomic differences among cells types (non-genomic equivalence)

•Differential transcription

•Differential nuclear RNA processing

•Selective messenger RNA translation

•Differential protein modification

I. Genomic Differences

•Weismann proposed only germ cells contained complete genome

•Somatic cell differences occur by receiving specific “determinants”

•So genomes of different cell types would be different

Weismann’s Germ Plasm Theory

I. Genomic Differences

•Earlier saw evidence for “genomic equivalence”

•Is there any evidence for genomic differences (genomic nonequivalence) among cell types?

I A. Gene Loss

Ascaris (Parascaris)

•All somatic cells undergo chromosome diminution (loss of part of chromosome)

•About 27% of DNA lost in somatic cells of Ascaris, 85% in Parascaris

•Only vegetal-most cells (with germ plasm) retain all of chromosomes  germ cells

Gall Midges (Wachtiella)

•At 16-nuclei stage, 2 nuclei enter germ plasm at one pole

•These 2 nuclei retain all 40 chromosomes  germ cells

•Other 14 nuclei lose 32 of 40 chromosomes  somatic cells

The fly Sciara

•Sperm haploid, 2 maternal X chromosomes

•Egg haploid, 1 X

•Zygote: 2 sets autosomes, 3 X chromosomes

During development of somatic cells

•Males: both paternal (sperm-derived) X’s lost; both autosome sets retained (2A/XO)

•Females: One paternal (sperm-derived) X lost; both autosome sets retained (2A/XX)

•Males XO, females XX in somatic cells

During development of germinal cells

•Males: One paternal (sperm-derived) X lost; both autosome sets retained (2A/XX)

•Females: One paternal (sperm-derived) X lost; both autosome sets retained (2A/XX)

•Both male and female germ cells XX

The Bandicoot, a marsupial

•XX/XY sex-determining system

•Germ cells retain XX or XY genotype

•However, in somatic cells

–Females lose paternal X, so cells XO

–Males lose Y (paternal), so cells XO

So, patterns seen with gene loss:

•Somatic versus germinal determination

–Parascaris

–Wachtiella

–Bandicoot

•Sex determination

–Sciara

•Weismann was correct for some special cases!

•If chromosome loss limited to maternal or paternal, always paternally-derived are lost

–Implies maternal and paternal chromosomes are “marked” differently

I B. Gene Inactivation

•Keep all of chromosomes, but change DNA so DNA can’t be expressed

•In mealy bugs (Homoptera), sex determination is haplo-diploidy (haploid = male, diploid = female)

•However, cells of males do contain both sets of chromosomes

•Paternally-derived set present butinactivated in somatic cells of males

•Sperm contain only maternally-derived chromosome set

•Both paternal and maternal sets active in somatic and germinal cells of females

•We will look at another example later with X-chromosome inactivation in mammals

I C. Gene Amplification

•Increase number of copies of specific genes in relation to other genes

•Increase restricted to certain cell types

rRNA genes

•18S and 28S rRNA genes in amphibian oocyte

•Prior to amplification about 500 copies per haploid genome

•Located in NOR

•Each repeat amplified 1,000 times

•After amplification are 500,000 copies per haploid genome

•Extra genes located in extra nucleoli

•Similar rRNA amplification in oocytes of fish, molluscs, insects

Chorion genes in Drosophila

•Ovarian follicle cells amplify genes for chorion proteins about 160 times

•Chorion proteins added to eggs

•What do the examples of amplification have in common?

•Large quantity of product needed in short time period

II. Differential Transcription

•Gene Structure

•Transcription Factors

•Silencers

•DNA Methylation and Gene Activity

II A. Gene Structure

•DNA bound to histone proteins = chromatin

•Nucleosome

–Octet of histone proteins

–140 bp DNA wrapped around histone core

–50 bp DNA between nucleosomes

• “Beads on a string” structure

•“Beads on a string” structure folded into solenoid via interaction with histone H1

•DNA in this configuration is transcriptionally inactive

•Thus, most native DNA is “repressed”

•Promoter region

–RNA polymerase binding, transcription factor binding

–“upstream” (- bases)

•Transcription Initiation Site

–Where RNA polymerase begins transcription

–+1 location

•Translation Initiation Site

–ATG in DNA (AUG in mRNA)

–Where ribosome will begin translating to add amino acids

•Translation Initiation Site

–Gap between transcription & translation initiation site the 5’ leader sequence (untranslated region) on mRNA

•Coding Region

–Composed of exons and introns

–Exons code for amino acids

–Introns do not code for amino acids (introns removed)

•Translation Termination Codon

–Signal to ribosome to stop protein synthesis

–UAA, UAG, UGA on mRNA

•3’ Untranslated region

–Transcribed but NOT translated

–Has poly-A tail attached after transcription

II A. Gene Structure

Promoter region

•TATA box –30 bases upstream from transcription initiation site

•RNA polymerase binds to TATA box

•Assembly of RNA polymerase requires interaction with transcription factors

•Transcription factors are proteins from other genes

•TBP (TATA-Binding Protein) on TATA

•TBP stabilized by interactions with TAF (TBP-associated factors) on upstream promoter regions

•SP1, NTF-1, 250, 110, 150 are all TAF’s

Enhancers

•DNA sequences “cis” to promoter

•Distant from promoter (up to 50 kb)

•May be at 5’ end, at 3’ end, in introns

•Interact physically with promoter to increase transcription

II B. Transcription Factors

•Proteins that bind to promoter or enhancers

•Can increase or decrease transcription rates

•Can be grouped in “families” based on structural similarity

•Helix – Loop – Helix

Each factor has up to 3 major domains

•DNA-binding domain

–Binds to specific DNA sequence

•Trans-activating domain

–Activates/suppresses gene transcription

•Protein-protein interaction domain

–May be present, interacts with other transcription factors

II C. Silencers

•DNA regions that actively repress transcription

•“negative enhancers”

•Example includes Neural Restrictive Silencer Element (NRSE)

•Binding of zinc finger transcription factor NRSF (neural restrictive silencer factor) to NRSE blocks gene expression

•NRSF binds to NRSE in non-neural tissue to block neural-specific gene expression

Figure 5.17 Silencers

Two groups of transgenic mice embryos

•“A” transgene with lacZ and NRSE silencer

•“B” transgene with lacZ and NO silencer

•lacZ codes for â-galactosidase

•Examined expression of â-galactosidase

Figure 5.17 Silencers

•Blue stain indicates presence of â-galactosidase

•“A” (w/NRSE) only expresses â-galactosidase in neural tissue

•NRSE repressed enzyme in non-neural tissue

Figure 5.17 Silencers

•“B” (w/out NRSE) expresses â-galactosidase in ALL tissue

•NRSE absent so NO repression of enzyme in any tissue

•Thus, NRSE blocks expression in non-neural tissue

II D. DNA Methylation & Gene Activity

•In VERTEBRATES, differentiated cell DNA has specific methylation pattern

•Cytosine is methylated when in CpG backbone pairs

•Methylation patterns change during development

•Very early embryos have overall low levels of methylation

•As cells become determined and differentiate, methylation levels rise in a tissue-specific manner

•Low methylation allows gene expression

•Addition of methylation blocks gene expression

•Patterns of methylation maintained after mitosis by a “maintenance methylation” enzyme

Human β-globin gene complex of three genes

•ε in early embryo

•γ in later development

•β in adult

Pattern of promoter methylation changes to match changes in gene expression

•ε unmethylated in early (6 week) embryo, becomes methylated at 12 weeks

•γ methylated in early (6 week) embryo, becomes unmethylated at 12 weeks, then methylated late in embryogenesis

•β methylated until late in embryogenesis, then becomes unmethylated

Genomic Imprinting

•Marking DNA regions/chromosomes as maternal or paternal

•Most DNA is NOT imprinted as maternal or paternal

•Normal Mendelian patterns of inheritance and expression if NO imprinting

•At least 30 mammalian genes imprinted as maternal or paternal

•Imprinting pattern related to methylation differences (methylated NOT expressed)

•Methylation differences inherited from egg and sperm (genes “marked” in gametes)

•For these 30 genes, only ONE of two alleles is expressed

•Either maternal OR paternal allele expressed for each gene

•Maternal/paternal expression differs among imprinted genes

–For some genes, maternal expressed

–For other genes, paternal expressed

•Thus, BOTH maternal and paternal genomes necessary for development

•Only PATERNAL Igf2 gene (Insulin-like growth factor 2) expressed in mice

•Only MATERNAL Ifg2r (Igf2 receptor) gene expressed in mice

–Deletion of paternal Ifg2r gene has no effect on development

–Deletion of maternal Ifgr2r gene kills embryo

•In humans, deletion in specific region of chromosome 15 (46, del 15q11-q13) causes EITHER Prader-Willi OR Angelman syndrome

Prader-Willi (right)

•Mild mental retardation

•Obesity

•Small gonads

•Short stature

Angelman (left)

•Severe mental retardation

•Seizures

•Lack of speech

•Inappropriate laughter

•If deletion in PATERNAL region, result is Prader-Willi syndrome

–Only maternal gene functional

•If deletion in MATERNAL region, result is Angelman syndrome

–Only paternal gene functional

Figure 5.20 Inheritance Patterns for Prader-Willi and Angelman Syndrome

•Methylation differences are used to inactivate the X chromosome in mammalian dosage compensation

•In dosage compensation, all X’s but one per cell are inactivated

•Inactivated X’s are highly methylated; active X is hypomethylated

Figure 5.21(1) Inactivation of a Single X Chromosome in Mammalian XX Cells

•Barr bodies are inactivated X’s

•XX cell on left

•XXX cell on right

•In very early embryo, both X’s of XX are active

•Inactivation via methylation occurs in blastocyst stage

•In germ cells inactive X is reactivated

•Random inactivation in most species

•In mouse trophoblast (which gives rise to chorion), the paternal X is inactivated

•Blue where paternal X active (from transgene)

•Mouse day 4 blastocyst

•All cells show blue, showing paternal X active (both X’s active)

•ICM & trophoblast have both X’s active

•Mouse day 6 embryo

•Trophoblast cells (above) all pink, no blue

•Thus, all paternal X’s inactivated in trophoblast cells

•Note some ICM cells still blue

Even in the Barr bodies some genes are active

•Steroid sulfatase

•Genes for ovarian functioning

Classic example of Barr body/dosage compensation effects in calico cat coat color

•Patterning of black and orange color

•X-linked gene

•Heterozygote has one allele for black, one for orange

•Depending upon which X is active, coat color is either black or orange

Figure 5.22 X Chromosome Inactivation in Mammals

•Calico cats typically female

•Male calico cats occur, but rarely

•What is genetic basis for calico pattern in males?

III. Differential nuclear RNA Processing

•Initial transcript is NOT messenger RNA

•Initial transcript much larger than mRNA

•Initial transcript “nuclear” RNA (hnRNA or pre-mRNA)

Are two ways nRNA can be differentially processed

•nRNA selection

•nRNA differential (alternative) splicing

III A. nRNA selection Figure 5.25 Roles of Differential RNA Processing During Development

•nRNA transcribed in all cells

•Specific nRNA degraded in one cell type, but processed to mRNA in second cell type

Figure 5.25 Roles of Differential RNA Processing During Development

•Cell type 1degrades a and b

•Cell type makes c, d, e mRNA

•Cell type 2 degrades d and e

•Cell type 2 makes a, b, c mRNA

•Sea urchin CyIIIa mRNA found in ectoderm (codes for actin)

•Shown is gastrula shape of embryo

•Autoradiogram of binding of probe to CyIIIa mRNA

•Note probe binds only to outer cells (ectoderm)

•However, probe of CyIIIa intron and exon DOES bind to nRNA from endoderm and mesoderm

•Thus, CyIIIa transcribed in all germ layers

•Transcript results in mRNA only in ectoderm

III B. nRNA Differential Splicing Figure 5.25 Roles of Differential RNA Processing During Development

•Selective removal and retention of different exons results in different mRNA’s

•Splice points between exons and introns have consensus sequences

•5’Exon-3’Intron

AG-GU

•5’Intron-3’Exon

YnAG-G (Y = pyrimidine)

•Spliceosomes bind to splice point and cut RNA

•Spliceosomes contain snRNA’s and proteins

•Production of different spliceosome splicing factors in different cells “allows” differential splicing in different cells  different mRNAs

Figure 5.27 Alternative nRNA Splicing

•α-tropomyosin exists in seven different primary structures

•Only ONE α-tropomyosin gene, containing 11 exons

•Differential processing of initial transcript allows different α-tropomyosin in skeletal muscle, smooth muscle, fibroblast, liver, and brain

Figure 5.28 Alternative RNA Splicing to Form a Family of Rat -Tropomyosin Proteins

•About 30,000 structural genes in human genome

•One-third estimated to be differentially spliced

•Drosophila Dscam gene has 24 exons

•Over 10,000 different Dscam proteins found

IV. Selective messenger RNA Translation

At least three mechanisms

•Differential mRNA longevity

•Selective translation inhibition of mRNA

•Cytoplasmic localization

IV A. Differential mRNA Longevity

•Longer the half-life, greater the quantity of translated products

•Stability influenced by length of polyA tail (direct relationship)

•Casein mRNA has half-life 1.1 hours in rat mammary gland tissue

•During lactation, in presence of hormone prolactin, half-life increasedOo to 28.5 hours

•Thus, during lactation, amount of casein protein produced for milk increases

Figure 5.31 Degradation of Casein mRNA in the Presence and Absence of Prolactin

IV B. Selective Translation Inhibition of mRNA

•mRNA’s may be stored in inactive state

•Biochemical changes activates translation

•Oocytes have large diversity of stored mRNA that are NOT translated until after fertilization

•5’ cap and 3’ untranslated region (UTR) important in regulating translation

•No cap or no poly-A tail, no translation

•Adding cap or adding tail may allow translation

•Binding of inhibitor to 3’UTR prevents translation

•In amphibians, maskin protein binds 5’ and 3’ ends together to prevent translation

IV C. Cytoplasmic Localization

•Segregate mRNA in particular cytoplasmic regions of oocytes

•Translation thus results in either gradients of products or localization of products in specific blastomeres

•Vg1 mRNA in vegetal portion Xenopus oocyte

V. Differential Protein Modification

•Posttranslational modification of polypeptide

•Insulin made by removing sections of precursor polypeptide

•ER may add “address tag” to polypeptide to specify destination

Last updated 19 January 2004

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