sequencing of the human -> functional genomics

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• sequencing of the human -gt functional genomics
• Gene-expression microarrays and RNA interferences
  (RNAi)
• ATM/NF?B and ATM/p53-mediated arms

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functional genomics

• to gaining system-level understanding of the
  mechanisms
• gene products interact and regulate each other
• physiological processes during normal development
  and in response to homeostatic challenges

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Gene-expression microarrays

• https//www.vbi.vt.edu/ article/articleview/145

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RNA interferences (RNAi)
(RNA-induced silencing complex)

• www.mpg.de/.../ EEB/200432_035.shtml

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RNA interferences (RNAi)

• www.life.uiuc.edu/ shapiro/RNAiApps.html

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ATM/p53 -mediatedATM/NFkB -mediated
G1 checkpoint
Ataxia- telangiectasia ( AT)

• www.biocarta.com/ pathfiles/m_atmPathway.asp

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NFkB
ATM/NFkB -mediated

• www.mbb.yale.edu/ fl/fl_s_ghosh.htm

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ATM
NFkB
ATM/NFkB -mediated

• http//www.emdbiosciences.com/popup/cbc/NFKB_Inter
  active_Pathway.htm

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Hypothesis

• the combined experimental strategy of expression
  arrays and RNAi is indeed a powerful method for
  the dissection of complex transcriptional
  networks, and that computational promoter
  analysis can provide a strong complementary means
  for assessing the accuracy of this dissection.

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Definition of the damage-responding gene set
Microarray analysis
??siRNA knocked-downcellular systems
Computational promoter analysis
GO functional gene annotations
Cluster analysis
TRANSFAC


- New candidate target genes


Database search

Adapted from Thomas Werner Biomolecular
Engineering, 17 87-94 (2001)
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??siRNA knocked-downcellular systems

• Materials and methods

DNA fragments
To be cloned
pSUPER retroviral vector
To be transfected
HEK293 cell (????)
(selected with puromycin or hygromycin)
??????siRNA??,?????????????????????????,??????????
?????????,??????????? ???????????siRNA??,????????
??????
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?Western blotting ?? RNAi

• RNAi????????????,????????????

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Sample preparation and microarray hybridization

• Materials and methods

isolated using TRIzol reagent treated with DNase
I phenol/chloroform extracted ethanol-precipitated
and quantitated.
RNA
HEK293 cell
(4 h with 200 ng/ml of NCS.)
Affymetrix Human Focus Gene- Chip arrays
10 ??? five cellular systems (uninfected and
the LacZ control cells and cells knocked-down for
Rel-A, p53 and ATM), each probed at two time
points without treatment and 4 h after exposure
to NCS.
(All samples were probed in independent
triplicates)
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Computation of gene expression levels from
microarray signals

• Materials and methods

RMA method
1. RMA ???, ?????? 2. RMA ???????????????
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Definition of the damage-responding gene set

• Materials and methods

DMA method ???at least 1.5-fold in one control
(either the uninfected or the LacZ-infected
cells), and at least 1.4-fold in the same
direction in the other control.
A total of 112 genes that were induced in both
controls met this criterion and are referred to
as the damage-induced gene set. Only seven genes
met an analogous criterion for repression in
response to NCS treatment
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Cluster analysis

• Materials and methods

112 gene ?? the EXPANDER package ??
average-linkage hierarchical clustering
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GO functional gene annotations

• Materials and methods

The gene ontology (GO) annotations
Computational promoter analysis

• Materials and methods

PRIMA software
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Quantitative real-time RT-PCR

• Materials and methods

real-time PCR
cDNA
Five micrograms of total RNA
oligo(dT)
SuperScript II RNase H- reverse transcriptase
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??

• RNAi and microarray technologies and a recently
  developed computational tool are powerful
• off-target effects
• computational promoter analysis was highly
  enriched for the binding signature of
  ATF2/ATF3/Jun

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??

• RNAi, microarrays and computational promoter
  analysis ?? dissection of transcriptional
  networks ???????
• Targeting the primary activator of a DNA damage
  response network, the upstream regulator(ATM) was
  indeed required for the induction of much of the
  network, the two downstream regulators
  (p53/NFkB)mediated the activation of largely
  disjoint sets of genes
• Statistical tests ?? computational promoter
  analysis ?????