L14 : C. elegans Gene Regulatory Networks that Control Ageing Flashcards
How does functional genomics contribute to understanding ageing?
Functional genomics uses high-throughput techniques to analyse large datasets from genome sequencing project
Unbiased approaches to assist in understanding how IIS and DAF-16 control ageing
What are some key techniques in functional genomics?
- RNA-mediated interference (RNAi)
- RNA profiling (eg. microarrays, RNA-seq)
- Chromatin profiling (eg. ChIP, DamID)
- Proteomics
- Metabolomics
How was RNAi used in C.e?
- Introduce dsRNA by microinjection, soaking, feeding
- Feed worms E. coli containing plasmid expressing dsRNA for gene X
- Feeding libraries prepared (E. coli strain for each gene in genome)
- Screen whole genome for gene where knockdown increases lifespan
What is the mechanism of RNA interference in gene silencing?
- Diver complex chops dsRNA into ~21 bp fragments called siRNAs (small interfering)
- siRNAs bind RISC (RNA-induced silencing complex)
- RISC uses siRNA as guide to find complementary mRNA and degrade it
- Silences gene expression for that siRNA
Note: System likely evolved as antiviral defence mechanism
What role does HSF-1 play in regulating ageing in C.e?
HSF-1 is TF that activates HSP genes
- Activated by trimerisation in response to stress
- RNAi knockdown of HSF-1 causes progeria (accelerated ageing)
- Overexpression of HSF-1 extends lifespan
- RNAi of HSD-1 suppresses daf-2 mutant longevity HSF-1 required for full lifespan extension seen in daf-2 mutants
Why is DNA microarray analysis used to study DAF-16 and ageing in C.e? Instead of mutational analysis?
Need unbiased method of screening for processes regulated by DAF-16
- Whole genome transcriptional profiling
Mutational analysis has failed to identify daf-16 regulated genes:
- Daf-16 may regulate many of the 20,000 gene genome, which act together to extend life
- Effects of individual genes may be small
What is a DNA microarray and how is it used?
Solid surface (glass, plastic, silicon) containing microscopic spots of DNA
Use: measure gene expression by detecting mRNA levels across many genes simultaneously
Method:
1. Isolate mRNA from organism and convert to cDNA
2. Labelled cDNA (eg. fluorescence) applied to microarray
3. cDNA binds to complementary DNA on chip
4. Fluorescence level indicates gene expression level
How can DNA microarrays be used to compare gene expression under different conditions?
DNA microarrays allow comparison of whole mRNA populations from two samples
- Comparing fluorescence intensity allows identification genes responsible for varied gene expression
Eg. Long-lived vs short-lived C.e
What are the main types of microarrays and differences?
- Spotted arrays
- Uses ~0.5 kb genomic DNA fragment probes
- Can suffer from cross-hybridisation (especially in multigene families) - Oligonucleotide arrays (GeneChips)
- Use unique short sequences (~25 nucleotides) per gene
- More specific and sensitive than spotted arrays - RNA-seq (direct mRNA sequencing)
- Not microarray but next-gen alternative
- Provides precise, quantitative, unbiased expression profiling
What is cluster analysis and how is it used in gene expression studies?
Method used to analyse microarray (or RNA-seq) data
- Helps determine whether genes are regulated individually or in groups
- Identifies synexpression groups (sets of genes that are co-regulated and show similar expression patterns
- Groups often participate in related biological processes
How was cluster analysis applied to large-scale microarray date in C.e?
- Researchers combined data from 533 microarray experiments across various conditions
- Identified 44 clusters (synexpression groups) - sets of co-regulated genes
- Visualised in 3D topographical map (each cluster = mountain)
What was discovered about gene regulation in long-lived daf-2 mutants?
McElwee and Murphy 2003
Used whole genome spotted microarrays for:
- daf-2 vs WT
- daf-2 vs daf-2/16 double mutants
- Identified hundreds of genes regulated (both up and down) by IIS
Used RNAi to test gene function:
- RNAi of upregulated often shortened lifespan -> potential longevity genes
How can researchers interpret long gene lists from microarray experiments?
Long gene lists can be overwhelming and prone to bias (‘fishing’)
Need unbiased statistical approach:
- Compare regulated gene list to known gene classes (eg. stress response)
- Are certain classes overrepresented?
- Calculate p-value indicating potential associations with lifespan
How were GeneChips used to identify DAF-16 target genes?
C.e whole genome oligonucleotide microarrays (GeneChips)
daf-2 (DAF-16 active) vs daf-2/daf-16 (DAF-16 inactive)
Identified genes with altered mRNA abundance due to DAF-16 activity
- 1348 genes upregulated
- 926 genes downregulated
- ~10% all genes showed DAF-16 dependent expression changes
Explain what hypothesis did overrepresentation analysis of gene clusters lead to and how can it be tested?
Long-lived worms
- Less collagen gene expression
- More dauer like molecular signature
- Implies longevity may involve partial activation of dauer specific programs
Hypothesis: daf-2 mutants have increased longevity due to partial activation of dauer specific mechanisms in adults
Perform unbiased analysis comparing gene expression lists in dauer larvae and daf-2 mutants
- Examine gene classes shared between both groups
- Shared enrichment suggests common mechanisms of lifespan extension
What gene classes are upregulated in dauers and daf-2 adults?
- smHSPs (heat shock proteins)
- Drug detoxification
- Cytochrome P450s (CYPs)
- Short chain dehydrogenases/ reductases (SDRs)
- UDP-glucuronosylytansferases (UGTs)
- Glutathione S-transferases (GSTs)
- These enzymes act to detoxify and render xenobiotic/endobiotic compounds soluble for excretion
What are the 2 phases of detoxification metabolism relevant to ageing theories?
Phase 1: Functionalisation
- Adds reactive functional groups to molecules
Phase 2: Conjugation
- Attaches solubility enhancing side groups, allowing excretion (urine, bile)
Pathways are upregulated in dauers and daf-2 mutants, suggesting role in longevity
What is the ‘Green theory of ageing’?
Proposes longevity is promoted by:
- Phase 1+2 detoxification systems
- HSP activity
- Somatic maintenance processes require a lot of energy to operate
Consistent with Kirkwood’s disposable soma theory:
- Organisms may cut costs on somatic maintenance
- Redeploy energy resources for reproduction
- Explains evolution of ageing
What evidence supports Phase 1+2 detox as longevity assurance mechanisms?
Overexpression of GST-10 (detox enzyme) in C.e leads to increased lifespan
Supports idea that enhanced detoxification helps extend life by maintaining cellular integrity
What is the model of evolutionary conservation in IIS-regulated ageing?
Signalling pathways (eg. IIS)
= public mechanisms = widely conserved
Longevity assurance processes (eg. detoxification)
= semi-public = conserved at process level, not always gene level (lack of orthology)
Damage accumulation and ageing mechanisms
= private = may be lineage specific
How is chromatin profiling (DamID) used to identify DAF-16 target genes?
Problem: array studies show too many genes regulated by DAF-16
Solution: Use DamID (DNA adenosine methyltransferase identification) chromatin profiling to find genes DAF-16 directly binds
- Cross reference genes DAF-16 binds with genes DAF-16 regulates
What was revealed on DAF-16 from DamID chromatin profiling?
- Most genes binding DAF-16 are not daf-16 regulated
- Identified 65 genes with high confidence
- Enriched for signalling genes (eg. IIS, EMP kinase, other TFs
Conclusion: DAF-16 appears to be regulator of regulators and does not directly regulate maintenance genes
