Biosci 755 Genomics and Gene Expression

9 sessions: 7, 14, 21 & 28 March, 4 April, 2, 9, 16 & 23 May.

LecturersAssociate Professor Brian Murray - course coordinatorAssociate Professor Rob YoungProfessor Russell SnellEach responsible for three sessions, the first will be an overview lecture of the general area and the second and third will be student seminars.Attendance at ALL sessions is compulsory.

•Each student will give ONE seminar.•These should be of c. 20 min duration and allow c. 10 min for questions from fellow students.•Seminars will be marked using a variety of criteria given on one of the following slides.•The seminar mark will make up 15% of the overall mark.•Seminar topics will be assigned today, see your handout. I expect a bit of background to also be provided in addition to the specfied paper you are presenting.•You also need to prepare a summary to give to the other members of the class. Hand in a hard copy at the resource centre at least 30 minutes before the class and they will copy it for you (need 25 copies).

• Each student will also write ONE essay.• Essay topics will be given out on 25 March.

Topics will be emailed to your University email address.

• The essay mark will make up 15% of the overall mark.

• Final examination will be of 3 hours duration in the normal examination period.

• Overall marks = One seminar (15%) and one essay (15%) (together make up in-course mark of30%) and final examination (70%).

Genome structure, organization and evolution - the wider perspective

Biosci 755 Genomics and Gene Expression

7, 14 & 21 March, 2011

Overview of five topics today

• Chromosome number and karyotype variation

• Genome size variation

• Ordering of chromosomes in the nucleus

• Heterochromatin

• Polyploidy as the key process in genome

evolution

One key question to keep in mind is, why are eukaryote genomes so variable in some aspects and constant in others?

Variability can be seen at many different levels and the aim of this first section of the course is to explore this variation and try to understand its basis and possible significance.

Is this variation just due to chance or can we see some causes or adaptive value in the variation?

• Two ways of looking at variation that can be seen as conflicting but are best thought of as being interrelated.

• Natural selection shapes phenotypes, remember that the genome is a phenotype just like any other aspect of the organism.

• Neutral theory of genetic variation where mutations have no obvious adaptive significance and stochastic (random,variable) processes result in the fixation of genetic differences.

Nature 470: 289-294

10 February 2011

• Most obvious feature when genomes are compared is that there are big differences in chromosome number, 2n = 2 to 2n = 1200.

• Genetic consequences are obvious in that the number of linkage groups changes - more chromosomes = more recombination.

• Changes in chromosome number are not random and in some groups all the species can have the same chromosome number and basic karyotype.

1. Chromosome number and karyotype variation

• Chromosome morphology or shape can also be highly variable.

– Some groups have highly conserved karyotypes whereas in others all the species have very different karyotypes.

– Examples of highly conserved karyotypes, that are easy to recognize in that they are bimodal, include the birds and the plant family Aloaceae.

– In many other groups almost anything goes.

Variation in chromosome number and size in the plant family Aloaceae but the karyotypes are always bimodal.

This family shows karyotypic orthoselection.

From: Brandham and Doherty (1998) Genome size variation in the Aloeaceae, an angiosperm family showing karyotypic orhtoselection. Annals of Botany 82 (Supplement A): 67-73.

Karyotypes of these three species from the Commelinaceae are highly diverse, in size, number and shape.

From: Jones and Colden (1972) Chromosomes and the classification of the Commelinaceae. Botanical Journal of the Linnean Society 65: 129-162.

How do differences in chromosome number arise?

• Polyploidy or whole genome doubling, to be discussed later.

• Robertsonian translocation. These are chromosome fission or fusion events where there is a loss or gain of centromeres without any overall change in gene content.

Can see how selection acts on karyotypes, neat experiments described in:Schubert I, Oud JL (1997) There is an upper limit of chromosome size for normal development of an organism. Cell 88: 515-520. New karyotypes produced by mutations. When chromosome arms become too long, they exceed the dimensions of the spindle and this results in an increased frequency of incomplete separation of the longest chromatid, resulting in cell death. In this way the chromosome complement is constrained = stabilizing selection.

• Genome size is also hugely variable e.g., 5000x in eukaryotes, 2500x in flowering plants (angiosperms).

• Is there an evolutionary trend?

2. Variation in genome size

Phylogenetic distribution of genome sizes in genera of the Liliaceae.

From: Leitch et al. (2007) Journal of Evolutionary Biology 20: 2296-2308.

• Raises the question, are genomes doomed to grow or are there complementary mechanisms to reduce or limit their size?

• Phylogenetic studies suggest that most basal groups are characterized by smallest genome sizes.

• But there are also many examples of more derived groups being characterized by having small genome sizes.

• Probably dealing with a dynamic situation and a key question is -

• Is there any evidence for an adaptive aspect to genome size variation? Is genome size correlated with gene number?– Clearly no, the number of genes in many (most) sequenced

plants and animals is remarkably similar. So what is the rest of the DNA made up of and what does it do?

– The death of the ‘junk DNA’ hypothesis?• Are we simply ignorant about the ‘activity’ of non-

protein coding sequences?• Three important points:

– At least 95% of human genome is transcribed but the genes make up only 1-2% of the genome.

– There are large blocks of highly conserved repeats in mammalian (and other) genomes. Highly suggestive that they have a function.

– Nucleotypic effects of DNA mass.

What are the key mechanisms that affect genome size identified by recent genome analyses ?

• Increase– Genome duplication

• Whole genomes via polyploidy• Segmental duplication

– Transposable element insertion and deletion• Retrotransposons• Transposons

– Hybridization– Horizontal transfer

• Decrease– Legitimate and illegitimate recombination between repeated

sequences

Growing chromosomes in Nicotiana hybrids and mouse cell cultures

Gene duplication in Arabidopsis

From: Paterson AH et al. (2000) Comparative genomics of plant chromosomes. The Plant Cell 12: 1523-1539.

Dot plot showing location of duplicate genes

Location of duplications on Arabidopsis chromosomes

From:Yu J et al. (2005) The genomes of Oryza sativa: A history of duplications. PLoS Biology 3: 266-281.

Duplicate segments in rice

Duplications can be dated by computing the number of substitutions per silent site (Ks), used here to identify segmental duplications in rice.

One pair of segments on chromosomes 11 and 12 result from a more recent segmental duplication event. They have fewer substitutions, the red bars on the graph.

From:Yu J et al. (2005) The genomes of Oryza sativa: A history of duplications. PLoS Biology 3: 266-281.

Transposable element activity - Retrotransposons

From: Wicker T et al. (2005) A detailed look at 7 million years of genome evolution in a 439kb contiguous sequence at the barley Hv-el4E locus: recombination, rearrangements and repeats. The Plant Journal 41: 184-194.

From: Griffiths et al. (2005) Introduction to Genetic Analysis, Freeman.

Molecular clock can be used to ‘date’ the time of insertion of LTR retrotransposons

In this example from maize can see that there have been many independent insertion events, some recent and others approximately 5.5 million years ago.Comparative studies of the same region in sorghum show that there are few retrotransposons - maize genome is approximately 3x larger than the sorghum one.

Model for the evolution of the Hv-elF4E locus in barley

From: Wicker T et al. (2005) A detailed look at 7 million years of genome evolution in a 439kb contiguous sequence at the barley Hv-el4E locus: recombination, rearrangements and repeats. The Plant Journal 41: 184-194.

Transposons are also important components of genomes

Sequence composition of genomic libraries and ESTs in the Triticum-Aegilops group, (a) Unfiltered Ae. tauschii shotgun library, retroelements comprise 53.5% and transposons 13.3%.(b) Methylation-filtered Ae. tauschii shotgun library, show an expected increase in non-repetitive sequences.(c) Transposable elements from the wheat (Triticum aestivum) EST database (TAGI) showing that many of the retrotransposon and transposon elements are active.

From: Li et al. (2004) Sequence composition, organization, and evolution of the core Triticeae genome. The Plant Journal 40: 500-511.

From: Lynch M (2007) The Origins of Genome Architecture, Sinauer.

Hybridization and genome size increase in Helianthus

•Hybrid species (all three derived from same parental species) show increased genome sizes compared to parents.•Newly synthesized hybrids and recently formed populations do not show an increase in genome size.•Populations of one of these show significant differences in genome size.

From: Baack E et al. (2005) Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species. New Phytologist

From: Martin W et al. (2002) Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. PNAS 99: 12246-12251.

The microbial origin of genes in the Arabidopsis genome

What about mechanisms of reduction?

Phylogenetic studies, as we have seen, show that small genomes can be found in organisms at both the base and tips of phylogenetic trees, so there must be mechanisms to counteract genome ‘growth’.Plants with genomes of 0.06pg/1C (c. 63 Mbp) have individual chromosomes that are the same size as a bacterial genome!

Mechanisms for genome reduction in Arabidopsis

From: Devos K et al. (2002) Genome size reduction through illegitimate recombination counteracts genome expansion in Arabidopsis. Genome Research 12: 1075-1079.

From: Devos K et al. (2002) Genome size reduction through illegitimate recombination counteracts genome expansion in Arabidopsis. Genome Research 12: 1075-1079.

Evidence for genome reduction in Arabidopsis

Other possible mechanisms to prevent the proliferation of retroelements

• RNA interference (RNAi), formation of double-stranded RNAs that are cut into small interfering RNAs by the enzyme dicer. Small fragments are then separated, bind to mRNAs and initiate a self-sustaining loop.

• Methylation of retroelements results in transcriptional silencing.

• Development of fluorescent in situ hybridization (FISH) has allowed the analysis of chromosome disposition and behaviour at interphase.

• Can show that there is order in the interphase nucleus.

• Chromosomes occupy specific regions or domains and the existence of order suggest that it is there for a particular reason.

• Chromosome position is highly conserved within groups such as the mammals.

• Even extends to specific chromosome segments that may have been translocated to different chromosomes.

3. Chromosome order in the nucleus

• Does nuclear architecture regulate gene expression?

• In F1 hybrids in plants there is spatial separation of whole genomes and this has been suggested to affect gene activity. Hybrids show the dominance of one parents characters.

• Does the position of a gene in the nucleus regulate its activity?

• Examples in human development and disease.• Chromosome Conformation Capture (3C)

effective technique for identifying interacting chromosome segments. Fix cells with formaldehyde to cross link interacting segments, digest away non-linked regions then sequence to identify linked regions.

Specific colocalization of genes on the same and different chromosomes. (a,b) Differences in positioning depends on whether genes are active or not driven at least in part through the shared colocalization with the same focal concentration of RNA polymerase II (Pol II), in a 'transcription factory’. (c) Association in naive mouse CD4+ T cells between the gene encoding the cytokine interferon γ (IFNγ) and specific sequences in the TH2 locus, including the genes encoding interleukin 5 (IL5) and the DNA-repair protein Rad50 as well as a DNase I hypersensitive site called RHS6 [5]. In this cell type, both gene loci are poised for rapid induction of low levels of expression.

Chuang & Belmont (2005) Genome Biology 6: 237

• Nucleolar organizer (rDNA) activity related to position both in the chromosome and the genome.

• Examples in Hordeum translocations where the movement of a NOR from one chromosome to another that already had a NOR results in the silencing of the translocated NOR.

• In Triticale, a wheat x rye hybrid the rye NORs are silenced and located peripherally but if plants are treated with a demethylating agent the inactive NORs are reactivated and located centrally in the nucleus.

4. Heterochromatin and euchromatin• What is heterochromatin?

– Regions of the chromosome/nucleus that stains more densely than the rest.

• Two types– Regions with a high density of repetitive DNA elements =

constitutive heterochromatin.– Developmentally regulated loci, regions or whole

chromosomes = facultative heterochromatin.– Can be identified in the chromosomes using differential

staining techniques.• How does it affect gene activity?

– Causes silencing of gene(s) e.g., X chromosome inactivation.

– Results from epigenetic changes in chromatin structure.– Represses recombination.

• Movement of genes into regions of heterochromatin or vice versa can cause gene silencing, but some genes are always located in heterochromatin.

• Complex interactions with a number of chromatin associated proteins. Recruit histone modifying enzymes (methyltransferases and deacetylases) that change to histone ‘tails’ and modify their structure (become more compacted).

• Followed by the recruitment of Heterochromatin Protein 1 (HP1/Swi6) which binds to the modified histones and allows heterochromatin to spread.

• Boundary elements prevent the uncontrolled spread of heterochromatin.

Mechanisms for the initiation of heterochromatin assembly.

Grewal and Jia Nature Reviews Genetics 8, 35–46 (January 2007) | doi:10.1038/nrg2008

Initiation by recognition of transcription factors (TF) or repetitive DNA, then recruit histone methyltransferase (HMT) and histone deacetylase (HDAC) that modify histone tails and change chromatin conformation. Boundary elements prevent the spread of heterochromatin.

• The control of transposable element activity has been suggested for the evolutionary benefit of heterochromatic silencing.

• Now know that heterochromatin formation is largely regulated by small RNAs and at least some of these originate from retrotransposons.

• Heterochromatin is implicated with a wide range of genomic ‘activities’ through the recruitment of various effectors.

The multiple interactions of heterochromatin with chromosome structure, behaviour and gene activity

Grewal and Jia Nature Reviews Genetics 8, 35–46 (January 2007) | doi:10.1038/nrg2008

5. Polyploidy, the key process in genome evolution?

• Key role has been highlighted by the many genome sequencing projects and the most unexpected organisms such as Arabidopsis thaliana are now known to have a polyploid ancestry.

• Remember that there are two main groups– Autopolyploids– Allopolyploids

• Has profound effects on a range of related nuclear characters.

• Neopolyploids and palaeopolyploids

From: Lynch M (2007) The Origins of Genome Architecture, Sinauer.

From: KL Adams & JF Wendel (2005) Polyploidy and genome evolution in plants. Current Opinion in Plant Biology 8: 135-141.

Inferred polyploidy events during the evolution of angiosperms.

Polyploidy has the potential to double the number of genes in a genome.Number of genes show interesting relationships•Yeast ca. 7000•Drosophila ca. 14000•Ciona ca. 16000•Human/mouse ca. 25000•Arabidopsis ca. 25500

Number of common or shared genes is high.Conservation of gene order or synteny is common.How does gene number relate to genome evolution?

Duplicate genes can either be preserved or lost

• Mechanisms for preservation- neofunctionalization - duplicates acquire new

functions. - subfunctionalization - duplicates acquire

complementary functions.• Loss of function mutations can eventually lead

to the loss of genes. Good example in yeasts that can be shown to be ancient tetraploids yet have only c. 500 more genes than their diploid ancestors.

• Meiotic effects, fertility and diploidization.– Multivalents or bivalents.

• Genome size– Cause a significant increase.– But polyploids commonly have smaller genome sizes

than expected. – Can show rapid loss of DNA on initial formation. But

this is not universal.– Changes are genome and chromosome specific.– Activation of retrotransposons.

• Does polyploidy contribute to genome separation?

•Genetic effects are complex and sometimes unpredictable. Can lead to gene silencing.

•Different alleles derived from the different parents expressed in different tissues.

•If polyploidy is ubiquitous, how do some organisms end up with only a few chromosomes? Robertsonian translocations.