Lecture 3 Part 1 DNA rearrangements Part 2 Epigenetics

Part 1 DNA rearrangements

2 broad consequences of DNA rearrangements

Creation of new genes, needed for expression in particular circumstances, as in the case of the immunoglobulins As a genetic switch for the regulation of gene expression

As a genetic switch in Salmonella

Switching of flagellar proteins (phase variation) enables the pathogen to evade its host's immune system

 Frequency of phase variation- ~ 1: 1000 cell divisions

When H2 flagellin is expressed, H1 is repressed (= phase 2 phenotype) When H2 is inactive, H1 is de-repressed (= phase 1 phenotype)

Mechanism involves inversion of H segment by sitespecific recombination

H segment is flanked by two inverted repeats that contain breakage sites used in recombination. H segment also codes for an enzyme(s) for the site-specific recombination (1) recombinase or "invertase" called Hin (H inversion) (2) at least one host function because inversion can still occur in crude cell extracts that don't contain recombinase
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(H segment)

Phase variation
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Use of conservative site-specific recombination to regulate gene expression in transgenic animals

Introduce transgene into a mouse harbouring a gene encoding site-specific recombination enzyme Induction of recombination enzyme results in activation of transgene expression Cre-LoxP system

Inactive gene Active gene

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Tissue-specific gene regulation

GENE OFF

GENE ON
Figure 5-79 Molecular Biology of the Cell ( Garland Science 2008)

Cre-Lox Site-specific recombination

Cre is a bacteriophage P1 integrase which catalyzes site-specific recombination between loxP sites (short direct repeats) This recombination also works in mammalian cells in vitro and in vivo

Yeast Mating Type Switching

S. cerevisiae can propagate in either the haploid or diploid condition
Haploid spore fuse (mate) to produce diploids Diploids undergo meiosis to produce haploid spores (sporulation)

Mating type:
Yeast of a given mating type secrete a small polypeptide that binds to a receptor on cells of the opposite mating type
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MATa Cell type Mating Sporulation Pheromone Receptor a Yes No a factor

MAT yes No factor

MATa/MAT a/ No Yes None none

Binds factor Binds a factor

Mating occurs only when the haploids are of different genetic types Sporulation occurs in heterozygous diploids which are capable of generating recombinants
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 Pheromone secreted by one cell type interacts with the receptor on another cell type When either receptor is activated, it interacts with the same G protein, which triggers a cascade reaction resulting in the activation of genes needed for mating Some yeast strains can switch their mating types (to facilitate formation of diploids)
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HML

Active locus MATa or MAT

HMRa

Switching occurs if MATa is replaced by HML , or MAT is replaced by HMRa (non-recipropcal exchange)
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Mechanism
Site-specific recombination initiated by a double strand break made at the MAT locus by the HO endonuclease Involves replicative transposition and gene conversion in which the recipient site (MAT) acquires the sequence of the donor type (HML or HMR) Only the MAT locus but not the HML or HMR loci is targeted by the HO endonuclease Results in unidirectional (nonreciprocal) switching
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Core region Y

SILENT

ACTIVE

SILENT

HML
frequent

MAT a

Change of mating type occurs when a replaces cassette

HMR a Change of mating type occurs when replaces a cassette a

frequent

No change of mating type rare occurs when cassette of same type replaces active cassette a

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 MAT locus controls the expression of pheromone and receptor genes involved in mating MATa expresses a1 protein constitutively Expression of 1 and 2 by MAT is inducible only in cells
1 protein turns on -specific genes needed for mating 2 protein turns off a-specific genes in haploids and represses a mating functions in diploids

a and proteins control transcription of various target genes Silent cassettes, HML and HMRa, are not expressed
Core region Y

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Haploid cells express a set of haploidspecific genes (hSG) together with aSG or SG Mat2 is a repressor

Mat1 is a transcriptional activator

In diploids, Mata1 acts together with Mat1 to turn off mating genes
Figure 7-65 Molecular Biology of the Cell ( Garland Science 2008)

Silencers repress expression at inactive cassettes

SIR = silent information regulators
HML MATa HMRa

SIR repression
HML

a1 1
MAT

SIR repression
HMRa

HML and HMR are regulated by silencers which lie outside these cassettes Silencers behave like negative enhancers works in either orientation and from a distance Egs. of proteins that act on silencers are SIR proteins which are believed to interact with histones to form heterochromatic (inert) structures
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Consequences of DNA rearrangements

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Recombination between direct repeats results in insertion, gene amplification and deletions

1. Insertion

z y x

2. Duplication (amplification)

z y x

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3. Deletion

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Recombination between inverted repeats results in inversions

Inversion

Inversion and deletion may result from both general homologous recombination as well as site-specific recombination The insertion and excision of bacteriophage from bacterial genome is an example of site-specific recombination
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DNA rearrangements due to Transposition

Transposition is responsible for much genetic rearrangement Homologous recombination between copies of transposons promote inversions, deletions, and rearrangements of host DNA

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Mutagenic effects of transposition on the function and expression of the target gene
Transposable element Exons Untranslated region

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Insertion of a transposon, which contain strong promoters, may alter level of gene expression:
Low gene expression Transposon with strong promoter, e.g. IS10R High gene expression

Weak promoter

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Insertion of a transposon may have a polar effect on the transcription of a polycistronic operon

Gene 1 Promoter

Polycistronic mRNA transcript Transposon or DNA fragment containing transcription termination sequence Gene 2 Shortened transcript

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Reading
Alberts et al Mol Biol of the Cell, 5th Ed
Chapter 5 pages 323-6 Chapter 7 pages 454-8

Lewin, B. Genes VII

p415-432, 437-441 (general recombination) P457-464, 467-470 (transposition mechanism) p485-489, 500-503 (retroviruses & retrotransposons) p508-519 (yeast mating type switching)
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Part 2 Epigenetics

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 Human chromosomes also carry a lot of information that is epigenetic, and not contained in the sequence of the DNA itself
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Epigenetic control of gene expression

1. Chromatin modification
Chromatin remodeling Covalent modification of histone tails

2. Position effect 3. Dosage compensation 4. DNA methylation & genome imprinting

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2. Position effects
Heterochromatin are transcriptionally silent and usually do not contain genes Activity of a gene depends on its position on the chromosome Many position effects exhibit an additional effect called position effect variegation
Heterochromatin may spread and retract (at low frequencies) The state of eu- or hetero- chromatin tends to be stably inherited in daughter cells
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Position effect variegation (PEV)

When in proximity to the telomere, ADE2 gene may be silenced by the spreading of adjacent heterochromatin, resulting in the accumulation of a red pigment White sectors refer to cells which have become reactivated Active white gene produces redeyed flies. White gene is silenced when moved next to heterochromatin Red patches in the white eye showed that the heterochromatin did not spread across this gene when the heterochromatin was formed during early development

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Cause of PEV in Drosophila

white

red
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Epigenetic control of gene expression

1. Chromatin modification
Chromatin remodeling Covalent modification of histone tails

2. Position effect 3. Dosage compensation 4. DNA methylation & genome imprinting

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Dosage compensation
Prevents excessive expression of X-linked genes in humans and other mammals Initial choice of which X to inactivate may be random (placental mammals) or always the paternal X chromosome (marsupials). X inactivation occurs in every somatic cell early during embryogenesis Once Xm or Xp is inactivated, that inactive state is faithfully maintained throughout subsequent cell divisions
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The calico cat is a female cat whose two X chromosomes specify a different coat colour (White patches are due to another gene)

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 Calico cats are always heterozygous females X-inactivation occurs in 64cell embryos - one of each pair of X chromosomes and its genes are randomly silenced Daughter cells inherit active or inactive Xm or Xp chromosomes, creating a cat with patches of coat color

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 Barr bodies are inactivated X chromosomes Observed as darklystained bodies in interphase nerve cells of female cats

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Barr bodies also observed in cells from humans with sex chromosome syndromes
46, X Y 45, X (Turners) 47, X X X 48, X X X Y Klinefelters syndrome 48, X X X X 49, X X X X Y Klinefelters syndrome 46, X X 47, X X Y Klinefelters syndrome

All, except one, of the X chromosomes appear inactivated and can be seen as Barr bodies
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Anhidrotic ectoderma dysplasia

Heterozygous female

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Dosage compensation in Drosophila

Females have 2X and males have 1X Mechanism of dosage compensation is different from that of mammals No X-inactivation in females, but male X-linked genes are transcribed at twice the level

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transcripts

transcripts

X X

X Y

X X

X Y

Female

Male

Female

Male

Dosage compensation in mammals

Dosage compensation in Drosophila

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Mechanism of X-inactivation

X-inactivation specific transcript (XIST) RNA spreads from XIC to cover the entire chromosome resulting in gene silencing

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Mechanism of X-inactivation
The X-chromosome heterochromatin is characterized by XIST RNA Variant histone 2A Hypoacetylated histones H3 & H4 Methylation of H3 Methylation of DNA (e.g. CpG islands found within many promoters)

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XIC

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Epigenetic control of gene expression

1. Chromatin modification
Chromatin remodeling Covalent modification of histone tails

2. Position effect 3. Dosage compensation 4. DNA methylation & genome imprinting

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4. DNA methylation and genome imprinting

C5 of cytosine in CpG-is usually methylated 60-90% of all CpG sequences in the genome are methylated Pattern of methylation is inherited by daughter strands during replication 5 mCpG 3 3 GpmC 5

Hypermethylation shuts down gene expression

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 Changes in pigmentation of mouse pup fur is directly linked to alteration in DNA methylation which can also be induced by feeding the pregnant mouse with genistein (phytoestrogen). The induced change also appear to protect the mouse offspring against obesity in adulthood
[Jirtle et al, Environ Health Perspectives, Apr 06]

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MBD=methyl-binding proteins

(a) Genome of higher eukaryotes has most DNA methylated except for CpG islands. (b) Methylated DNA bind MBDs that recruits enzymes that induce histone-tail deacetylation (HDAC) or methylation (c) Methylation pattern is erased during preimplantation embryogenesis and reestablished during implantation.

Mantenance methylase which recognizes hemi-methylated CpG preserves the pattern 51 after each cell division

DNA methylation patterns are faithfully inherited

1. Maintenance DNA methyltransferase (DNMT1)
Highly active on hemi-methylated DNA Adds CH3 to C in CG sequences that are already basepaired with a methylated CG

2. de novo methylase (DNMT3a, DNMT3b)

Add CH3 group to completely unmethylated CpG bp, thus creating, first a new hemi-methylated and then, full-methylated CpG
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Maintenance methyltransferases act preferentially on hemi-methylated CG residues

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 DNA methylation works together with other proteins to ensure that genes that have been turned off remain completely OFF Multiple mechanisms contribute to stable gene repression

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Genome imprinting

Mule horse x donkey

The mule is superior to the hinny and horse in strength, endurance, intelligence and disease resistance.

Hinny donkey x horse

Shorter ears, thicker mane and tail, stronger legs Certain maternally and paternally supplied genes are differentially expressed in mammals

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Genomic Imprinting
restricted to placental mammals and genes involved in fetal development occurs early - during formation of germ cells Imprinted genes are methylated and are not affected by wave of demethylation following fertilization

Imprinted allele inherited from the father

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 Methylation of genes in male gametes (this eg.) is reestablished soon after such that the diploid zygote carry 1 expressed allele (from mother) & 1 methylated allele (from father)

Imprinted allele inherited from the father

Majority of the mouse genes are NOT imprinted and would thus be inherited according to Mendelian laws

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E.g. Gene encoding insulin-like growth factor-2

Maternal copy NOT transcribed because of active CTCF insulator element Mouse with mutated maternal Igf-2 is normal

Paternal copy of Igf-2 is expressed Mouse with mutated paternal Igf-2 is stunted

Insulator element is methylated in the paternal allele

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Genomic Imprinting

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Imprinting and human disease

During normal fetal development, certain genes on chromosome 15 are suppressed on maternal chromosome and are expressed on paternal chromosome.

Maternally inherited chrom 15 deletion Paternally inherited chrom 15 deletion (paternal uniparental disomy) (maternal uniparental disomy.) - Angelman syndrome 60 - Prader-Willi syndrome

Reading
Alberts et al, 5th Ed, 2008 Chapt 4 p215-33 Chapt 7, p 467-77 Lewin, B, Genes VII, p 666-681 http://www.sciencemag.org/feature/plus/sfg/rese arch/rsch_epigenetics.shtml

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