Transcript Dissection of a DNA-damage-induced transcriptional network using

生科4甲 蔡德峰
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前言
• sequencing of the human -> functional
genomics
• Gene-expression microarrays and RNA
interferences (RNAi)
• ATM/NFκB 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
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•
https://www.vbi.vt.edu/ article/articleview/145
RNA interferences (RNAi)
(RNA-induced silencing complex)
•
www.mpg.de/.../ EEB/200432_035.shtml
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RNA interferences (RNAi)
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•
www.life.uiuc.edu/ shapiro/RNAiApps.html
G1 checkpoint
ATM/p53 mediated
ATM/NFkB
-mediated Ataxia- telangiectasia ( AT)
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•
www.biocarta.com/ pathfiles/m_atmPathway.asp
NFkB
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ATM/NFkB -mediated
www.mbb.yale.edu/ fl/fl_s_ghosh.htm
ATM
•
NFkB
http://www.emdbiosciences.com/popup/cbc/NFKB_Interactive_Pathway.htm
ATM/NFkB -mediated
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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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實驗流程圖
Microarray
analysis
建立siRNA knocked-down
cellular systems
Computational
promoter analysis
TRANSFAC
GO functional gene
annotations
Cluster analysis
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Database search
Definition of the damageresponding gene set
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*- New candidate
target genes
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Adapted from Thomas Werner Biomolecular Engineering, 17: 87-94 (2001)
建立siRNA knocked-down
cellular systems
• Materials and methods
DNA fragments
To be cloned
pSUPER retroviral vector
To be transfected
HEK293 cell (哺乳動物)
(selected with
puromycin or hygromycin)
病毒載體用于siRNA表達，其優勢在于可以直接高效率感染細胞進行基因
沉默的研究，避免由于質粒轉染效率低而帶來的種種不便,而且轉染效果更
加穩定。
最適用于：已知一個有效的siRNA序列，需要維持較長時間的基因沉默。
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以Western blotting 檢驗 RNAi
•
RNAi并不能完全阻斷基因的表達，特別是表達異常高的基因。
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Sample preparation and
microarray hybridization
• Materials and methods
HEK293 cell
(4 h with 200
ng/ml of NCS.)
RNA
isolated using TRIzol reagent
treated with DNase I
phenol/chloroform extracted
ethanol-precipitated and quantitated.
Affymetrix Human Focus GeneChip arrays
10 種狀態 : 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.14
(All samples were probed in independent triplicates)
Computation of gene expression
levels from microarray signals
• Materials and methods
RMA method
1. RMA 計算後, 信號明顯增強
2. RMA 使用齊次多項式證明數據改進更好
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Definition of the damageresponding gene set
• Materials and methods
DMA method 取數值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 使用 the
EXPANDER package 去
做 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
cDNA
Five micrograms of total RNA
oligo(dT)
SuperScript II RNase H- reverse transcriptase
real-time PCR
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討論
• 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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結論
• RNAi, microarrays and computational promoter
analysis 對於 dissection of transcriptional
networks 的研究是有力的
• 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 聯合 computational promoter
analysis 是高精確的
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