A Functional Genomics Approach to Ageing

Question 1

What is functional genomics?

Answer 1

This is the use of the high-throughput approaches that have been made possible by advances in sequencing and computing technology. This includes the use of RNA-mediated interference, microarrays, chromatin profiling, proteomics and metabolomics.

Question 2

How can elegans be knocked down using siRNA?

Answer 2

Introduction of the dsRNA can be by soaking, microinjection or – most commonly – by feeding. C. elegans consumes E. coli as a nutrient source. By feeding the worm recombinant E. coli that are expressing the dsRNA from a plasmid the elegans can be inundated with the dsRNA.

Question 3

What does the elegans feeding siRNA exposure mechanism allow for?

Answer 3

This allows for preparation of frozen ‘feeding libraries’, which come with a huge number of E. coli colonies that are each a different strain containing a plasmid that allows knockout of one of the genes in the worm.

Having a library with a strain for every gene in the genome greatly simplifies the process of screening large numbers of genes for their effect on ageing.

Question 4

What ageing mutant did RNAi screening of elegans identify?

Answer 4

In 2002 it was shown that RNAi of Heat Shock Factor 1 causes progeria (accelerated ageing) in C. elegans.

Question 5

What is HSF1?

Answer 5

HSF1 is the transcription factor which responds to stresses such as temperature, chemical stress, heavy metal poisoning or physical pressure by trimerising, allowing it to bind to heat shock response elements and activate the stress response.

Question 6

What does overexpression of HSF1 in elegans cause and what is the implication here?

Answer 6

Overexpression of HSF1 did increase longevity. However, this effect was dependent on the action of wt daf-16, and likewise the life-prolonging effects of daf-2 mutation was shown to be dependent on the action of HSF1.

This may indicate that the genes that are important for longevity are co-regulated by the IIS signalling pathway and the HSF pathway.

Question 7

What is spotted array analysis?

Answer 7

These are mass produced slides containing spots of DNA complementary to each of the genes found in the target organism. By extracting the transcriptome of cells from two different strains and converting the mRNA to fluorescently labelled cDNA using reverse transcriptase. By tagging the cDNA from the two strains with different colours, the comparative differences in expression can be analysed by the colour and relative intensity of the spots.

Question 8

What is the disadvantage of spotted array analysis?

Answer 8

Spotted Array’ technology is now somewhat outdated. Many of the probes were long sequences of around 0.5kb which were too promiscuous, often capturing the cDNA of various genes with similar sequences such as those within the same gene family.

Question 9

How have the issues with spotted arrays been overcome?

Answer 9

To get around this cross-hybridisation problem, short (22bp) sequences were designed for each gene in the genome that would bind that transcript’s cDNA uniquely. This is known as a gene chip (or oligonucleotide array), and is much more sensitive than the spotted arrays.

However, these do still have the disadvantage of being very difficult and time-consuming to analyse, and must be outsourced to private companies for this

Question 10

What is a topomap?

Answer 10

Many genes are co-regulated in clusters as opposed to individually, and so can be classed in synexpression groups. Gene chip analysis can identify these groups and produce a topomap showing the clusters and their relative expression level.

Question 11

What does the elegans topomap show?

Answer 11

44 synexpression groups from data gleaned from 553 microarray analyses.

Question 12

What genes are affected by daf-16 and how? What does this show?

Answer 12

To discover this, spotted whole-genome microarrays were used to compare the gene expression of wild-type worms against daf-2 or age-1 mutants, and later daf-2 vs daf-2(-):daf-16(-) mutants. The IIS pathway was shown to be controlling hundreds of different genes.

This was confirmed when the Gems’ Lab refined these experiments using gene chip analysis, which found that around 10% of all the elegans genes were regulated in some way by daf-16. 1,348 were upregulated and 926 downregulated.

Question 13

How does Daf-16 affect its target genes and why is this important?

Answer 13

Some of these genes were upregulated and some were downregulated. Both the upregulated genes and the downregulated genes were shown to be involved in ageing when RNAi of the daf-16 upregulated ones was shown to shorten lifespan and overexpression of the daf-16 silenced genes extended it.

This shows that there are many proteins involved in the ageing process, whose cumulative effect contribute positively or negatively to longevity.

Question 14

What previously considered group of proteins was identified as a daf-16 target?

Answer 14

Although some SOD family genes were found amongst these, a link cannot be drawn since there is no reason to assume they have any more importance over the other thousands of genes other than the existing bias towards that theory.

Question 15

How were pathways with potential relevance to ageing identified from the database of daf-16 targets?

Answer 15

In order to analyse the data in an unbiased way, it is better to look at which gene categories or families are effected. The overlap between the daf-16 regulated genes and the genes involved in a certain process must be analysed for significance. A large overlap indicates that the process is likely to be involved with ageing control.

Question 16

What did analysis of the level of overlap between daf-16 targets and pathway genes show?

Answer 16

The original spotted array analysis (McElwee et al 2003) used this method on the genes involved in dauer formation and in collagen production, showing that increased lifespan was associated with decreased daf-16 downregulation of the dauer genes and upregulation of the collagen genes.

This supports the original hypothesis that daf-2(-) mutation leads to expression of dauer longevity genes in the adult.

Question 17

What did analysis of genes responsible for sub-processes within dauer formation demonstrate?

Answer 17

This is a way of identifying which of the dauer genes are involved in ageing, and did in fact implicate novel processes as being involved with longevity. Overlap was also found between these and heat shock genes.

The novel genes belonged to four classes:
• Cytochrome P450s (CYPs)
• Short chain dehydrogenases/reductases (SDRs)
• Glutathione S-transferases (GSTs)
• UDP-glucuronosyltransferases (UGTs)

All of these are involved in toxin metabolism, detoxifying toxic xenobiotic and endobiotic compounds, indicating that detoxification may be involvewd in ageing.

Question 18

How does detoxification occur?

Answer 18

Metabolism of harmful chemicals (broad-spectrum detoxification) occurs in two stages; functionalisation reactions and conjugative reactions.

Phase one is performed by CYPs and SDRs and involves the addition of reactive groups to the toxic compound, allowing phase two to occur in which the compound is conjugated to a large soluble molecule by UGTs or GSTs (glucuronyl or glutathionyl used respectively).

Question 19

What is Kirkwood’s Disposable Soma theory of ageing?

Answer 19

It is postulated that the compromises made when allocating the finite energy available to the cell lead to gradual deterioration as repair and protective mechanisms are not properly provided for.

This fits with the ‘green theory’ of ageing.

Question 20

Why does Kirkwood’s Disposable Soma theory of ageing fit with the green theory?

Answer 20

The huge variety of toxins that may threaten the cell (that may be xenobiotic of endobiotic) present an informatics challenge to the genome which could, for the cell, be the equivalent of a money pit.

Question 21

How was the green theory tested, and what were the results?

Answer 21

This can easily be tested by overexpression of the relevant genes, which was done in drosophila. Overexpression of GST-10 in drosophila was shown to cause an increase in detoxification of lipid peroxidation products and an increase in lifespan.

Similarly, overexpression of sniffer SDR in drosophila was shown to extend youthspan, but not lifespan.

Question 22

Why was drosophila Nrf-2 investigated in relation to the green theory?

Answer 22

In order to look at the more wide-scale effects, the expression of a transcription factor that controls expression of the detoxification phase-2 genes was analysed. Nrf-2 is known to activate this in response to oxidative or electrophic stress.

Question 23

How is Nrf-2 regulated?

Answer 23

Nrf-2 is inhibited by Keap1 by sequestration in the cytoplasm. Keap1 is sensitive to the redox state of the cell by a similar mechanism to thioredoxin, it contains sulphydryl groups that are close enough to form disulphide bridges of thioester bonds under oxidising conditions.

This causes it to release Nrf-2 allowing it to relocate to the nucleus, where it binds a small nuclear cofactor called Maf and activates the phase-2 detoxification genes.

Question 24

How was the effect of Nrf-2 modelled?

Answer 24

The effect of Nrf-2 can be analysed using the C. elegans homologue Skn-1. Overexpression of skn-1 does indeed increase lifespan. The longevity effects of daf-2 mutation have been shown to be partially dependent on skn-1, so IIS signalling may be responsible for somehow downregulating the phase-2 detoxification genes through this TF.

Question 25

What question does the conservation of the effect of IIS signalling on ageing in worms, flies and mice raise? How can this be answered?

Answer 25

What is not known is whether it controls ageing through the same processes in each species. To answer this, what is affected by IIS signalling can be compared in the three species in terms of both orthologous gene regulation and regulation of processes.

This is an attempt to determine whether the mechanisms that affect ageing in each of these model organisms is public (conserved) or private (found only in that species).

Question 26

What did comparison of the IIS targets in the different model organisms show?

Answer 26

This found conservation of several of the processes involved between the three species, however gene level conservation studies did not show evolutionary conservation of orthologous genes.

Question 27

What is the explanation of the lack of conservation in orthologous genes regulated by the IIS pathway in model organisms?

Answer 27

This is because in genes such as those involved in phase-2 detoxification, there is a low level of orthology. This is a response to the difference in the targets of detoxification between the different lineages, causing significant divergence between the genes. The substrate specificities of the GSTs regulated by the IIS pathway is yet unknown.

The conclusion that can be drawn from this is that while the regulators process (IIS signalling) is conserved/public, the longevity-assuring process (broad spectrum detoxification) is semi-public and the ageing mechanism itself is private, as the nature of the molecular damage is dependent on what toxins the IIS signalled detoxification enzymes are responsible for

Question 28

What is an alternative approach to looking at the targets of daf-16?

Answer 28

Identifying the daf-16 regulated genes using gene-chips led to the issue of looking for ageing related genes amongst the thousands that are regulated by daf-16. Another approach to be taken is mapping the topology of the regulatory network in order to refine the search.

Question 29

What models were proposed for the daf-16 regulatory network? How was this investigated?

Answer 29

Two models were proposed for the structure of the network, either daf-16 directly regulating many target proteins, or daf-16 regulating a small number of targets which in turn regulate their own number of targets in a variety of parallel cascades.

In order to analyse this DamID Chromatin Profiling was used to identify the binding sites of daf-16 in the elegans genome.

Question 30

What is DamID chromatin profiling and how was it used to investigate daf-16 regulatory network topology?

Answer 30

By conjugating DNA adenine methyltransferase to daf-16, methylation sites can be created only in the places where daf-16 binds. These are easy to identify as C. elegans does not otherwise methylate its DNA.

The methylated sites are identified by digestion of the extrated genomic DNA with Dpnl, which cleaves off the methylated sequences. PCR amplification and fluorescent labelling of the methylated DNA allows for visualisation of the genes on which daf-16 binds.

Question 31

What were the results of daf-16 DamID chromatin profiling?

Answer 31

Of the 842 genomic binding sites of daf-16, only 65 were associated with any change in expression. It is suggested that the other “putatitive” daf-16 targets are inactive ‘parking sites’ for daf-16.

However, the fact that 65 of the targets were known to be regulated by daf-16 indicates that the second model is correct, and the rest of the daf-16 regulatory targets are targeted by downstream regulators.

The genes directly targeted by daf-16 are rich in signalling proteins and transcription factors, including a large number of those known to be involved in ageing regulation.