Discoveries about aging using genomic methods'

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Discoveries about aging using genomic methods.

• AS300-003 Jim Lund

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Genomic studies of aging

• Studies that examine changes in every gene or
  protein.
• Comparison of
• Young/old
• Wild-type/long-lived
• Dividing/senescent cells.

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Why genomic studies?

• The genes/proteins expressed determine the state
  of the cell.
• Rather than test hypotheses about the role of
  particular genes or gene/gene interactions survey
  entire genome to identify all the genes involved
  in aspects of aging.
• Complete genome sequences made genomic methods
  practical.

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Genomic methods

• Gene Expression Assay
• Microarray Hybridization
• Serial Analysis of Gene Expression (SAGE)
• Promoter analysis
• Bioinformatic analysis to find transcription
  factor binding sites.
• Chromatin Immunoprecipitation/promoter microarray
  (ChIPchip)
• Protein analysis
• Proteomic methods to identify proteins expressed
  at higher levels in one sample compared to
  another.
• Methods to identify proteins in mixtures of
  hundreds of proteins.

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Genomic methods

• Studies that examine changes in every gene or
  protein.
• Genomic aging experiments--comparison of
• Young/old
• Wild-type/long-lived
• Dividing/senescent cells.

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Comparison of senescent cell and aging cell gene
expression
Included senescence studies Human Fibroblasts Hum
an Prostate epithelial cells Human Mammary
epithelial cells Mouse Thymus epithelial cells

• Included aging studies
• Human Skeletal muscle
• Human Frontal cortex of brain
• Human Kidney cortex and medulla
• Mouse Brain cerebellum/neocortex
• Mouse Heart
• Rat Hippocampus, brain

Wennmalm et al., 2005
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Robust transcriptome signatures of cellular
senescence and aging
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Overall similarities between aging and senescence
and across species

• Gene expression changes between young and old
  tissue samples in mouse and human well
  correlated.
• Gene expression changes between dividing and
  senescent samples in mouse and human well
  correlated.
• Gene expression changes in mouse aging and
  senescence very well correlated.
• Gene expression changes in human aging and
  senescence NOT well correlated.

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Gene Ontology categories consistently altered in
aging and senescence

• Mitochondria
• ATP biosynthesis
• ATPase activity
• Respiratory gaseous exchange
• Proton transport
• Hydrogen ion transporter activity
• Protein folding
• Protein folding
• Lysosome
• Endoplasmic reticulum
• Metalloendopeptidase activity
• RNA processing
• Spliceosome complex
• RNA binding
• Nuclear mRNA splicing via spliceosome

Other Mitotic sister chromatid
segregation Regulation of cell cycle Negative
regulation of cell proliferation
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Study identifies conserved DAF-16 tagets in C.
elegans and D. melanogaster

• DAF-16 and its mammalian homologs bind to an
  identical consensus DNA sequence (TTGTTTAC) in
  vitro.
• Identified DAF-16 transcriptional targets by
  searching for DAF-16 binding sites in the
  regulatory regions of genes in C. elegans and D.
  melanogaster.
• Found 17 genes that are orthologous between
  Drosophila and C. elegans and bear a DAF-16
  binding site within 1 kb of their start codons in
  both species.

Lee et al., 2003
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Seven DAF-16 target candidate genes is regulated
by daf-2/insulin-like signaling in a
daf-16dependent manner.
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Longevity after RNAi of DAF-16 transcriptional
targets.
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DAF-16 regulated genes and their phenotypes

• RNAi
• Gene mRNA in daf-2(-) inactivation
• life-span
• C08B11.8, glucosyltransferase nc
  nc
• C10G11.5 (pnk-1), Pantothenate kinase 5X
  Shortened
• C39F7.5, Cytochrome c heme binding site
  3X nc
• E04F6.2 - nc
• F14F4.3 (mrp-5), ABC transporter nc
  Extended
• F27C8.1, Amino acid transporter -
  nc
• F43G9.5, Subunit of pre-mRNA cleavage factor 1
  0.4X nc
• F52H3.5, Similar to yeast stress-induced protein
  7X nc
• F54D5.7, Acyl-CoA dehydrogenase nc
  nc
• K07B1.3, Mitochondrial carrier nc nc
  
• T20B3.1, Carnitate acyltransferase -
  nc
• T20B5.3, Hyaluronoglucosaminidase nc
  nc
• T21C12.2 (hpd-1) 0.5X Extended
• T23B12.4 nc nc
• Y106G6H.7, Mitochondrial energy transfer protein
  nc nc
• ZC506.3, Phosphatidylserine synthase 1 nc
  nc

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Study of gene expression regulated by DAF-16 in
C. elegans

• Used full genome DNA microarrays to compare gene
  expression between
• Wild-type worms
• daf-2
• daf-16
• Other long-lived insulin-like pathway mutants

Murphy et al., 2003
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Spotted DNA microarray
orf 1
orf 2
orf 3
1
2
3
3 cm
4
5
6
200 spots
7
8
9
2
40,000 dot/9 cm
or
Condition 1
Condition 2
gt All human genes
mRNA
mRNA
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Spotted DNA microarray
orf 1
orf 2
orf 3
1
3
3 cm
4
5
6
200 spots
7
9
2
40,000 dot/9 cm
or
Condition 1
Condition 2
Condition 2
gt All human genes
mRNA
mRNA
mRNA
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Spotted DNA microarray
orf 1
orf 2
orf 3
1
3
3
1
2
3 cm
4
5
6
6
200 spots
7
9
7
8
2
40,000 dot/9 cm
or
Condition 1
Condition 2
gt All human genes
mRNA
mRNA
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Spotted microarray image
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Genes with large expression changes
daf-2 RNAi daf-16 RNAi daf-2
pathway mutants
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Study focused on two classes of DAF-16 regulated
genes

• Authors used hierarchical clustering to classify
  genes.
• Two clusters of genes were of particularly
  interesting.
• Class 1 genes that were induced in DAF-2
  pathway mutants and in daf-2(RNAi) animals but
  repressed in daf-16(RNAi) daf-2(RNAi) animals
• These were candidates for genes that extend
  lifespan
• Class 2 genes that displayed the opposite
  expression profile.
• candidates for genes that limit lifespan.

Murphy et al., 2003
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Daf-16 target metabolic and lipid synthesis genes
affect lifespan
Metabolic genes Lipid synthesis
genes Expts done in a daf-2 strain (black).
daf-16 control is in purple.
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Daf-16 target oxidative stress genes affect
lifespan
Expts done in a daf-2 strain (black).
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Class 2 genes (limit lifespan)
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DAF-16 regulated genes and their phenotypes

• Class I (extend ls)
• Catalases, superoxide dismutase, oxidoreductases
• Heat shock proteins
• P450s (detoxification)
• Mitochondrial genes (energy production, etc)
• Proteases
• Antimicrobial genes
• Unknown genes
• Class II (limit ls)
• Signaling pathway genes
• Vitellogenin (yolk protein) genes
• Transporters
• Insulin
• Unknown genes

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DAF-16 targets a model
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Comparing genomic expression patterns across
species identifies shared transcriptional profile
in agingMcCarroll et al., 2004

• Compared gene expression profiles from in aging
  expts in
• C. Elegans
• D. Melanogaster
• Saccharomyces cerevisiae
• Homo sapiens

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Method for cross species comparisons

• Identify corresponding genes (orthologs).
• Find gene expression values for these
  correspnding genes
• Calculate correlateion between expression in the
  two species.

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Many gene classes similarly regulated during
aging in worm and fly