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生科 4 甲 蔡德峰

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前言• sequencing of the human -> functional gen

omics

• 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

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

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RNA interferences (RNAi)

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

(RNA-induced silencing complex)

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RNA interferences (RNAi)

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

7• www.biocarta.com/ pathfi les/m_atmPathway.asp

ATM/p53 -mediated

ATM/NFkB -mediated

G1 checkpoint

Ataxia- telangiectasia ( AT)

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www.mbb.yale.edu/ fl/fl_s_ghosh.htm

NFkB

ATM/NFkB -mediated

9• http://www.emdbiosciences.com/popup/cbc/NFKB_Interactive_Pathway.htm

ATM/NFkB -mediatedNFkB

ATM

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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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Microarrayanalysis

Database search

Computational promoter analysis

*- New candidate target genes

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*Adapted from Thomas Werner Biomolecular Engineering, 17: 87-94 (2001)

TRANSFAC

實驗流程圖

Definition of the damage-responding gene set

Cluster analysisGO functional gene annotations

建立 siRNA knocked-downcellular systems

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建立 siRNA knocked-downcellular systems

• Materials and methodsDNA fragments

To be transfected

To be cloned pSUPER retroviral vector

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

HEK293 cell

• Materials and methods

(4 h with 200 ng/ml of NCS.)

RNAisolated using TRIzol reagenttreated with DNase Iphenol/chloroform extractedethanol-precipitated and quantitated.

Affymetrix Human Focus Gene-Chip arrays

(All samples were probed in independent triplicates)

10 種狀態 : five cellular systems (uninfected and the LacZ control cells and cellsknocked-down for Rel-A, p53 and ATM), each probed at two time points: without treatment and 4 h after exposure to NCS.

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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 damage-responding 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 thiscriterion 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-linkagehierarchical 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

Five micrograms of total RNA cDNA

oligo(dT) SuperScript II RNase H- reverse transcriptase

real-time PCR

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討論• RNAi and microarray technologies and a r

ecently 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