生科 4 甲蔡德峰

生科4甲蔡德峰

前言 • sequencing of the human -> functional genomics • Gene-expression microarrays and RNA interferences (RNAi) • ATM/NFκB and ATM/p53-mediated arms

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

Gene-expression microarrays • https://www.vbi.vt.edu/ article/articleview/145

RNA interferences (RNAi) • www.mpg.de/.../ EEB/200432_035.shtml (RNA-induced silencing complex)

RNA interferences (RNAi) • www.life.uiuc.edu/ shapiro/RNAiApps.html

G1 checkpoint ATM/p53 -mediatedATM/NFkB -mediated • www.biocarta.com/ pathfiles/m_atmPathway.asp Ataxia- telangiectasia ( AT)

www.mbb.yale.edu/ fl/fl_s_ghosh.htm NFkB ATM/NFkB -mediated

ATM • http://www.emdbiosciences.com/popup/cbc/NFKB_Interactive_Pathway.htm NFkB ATM/NFkB -mediated

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.

實驗流程圖 Definition of the damage-responding gene set Microarray analysis 建立siRNA knocked-downcellularsystems Computational promoteranalysis GO functional gene annotations Cluster analysis TRANSFAC * * *- New candidate target genes * * * Database search Adapted from Thomas Werner Biomolecular Engineering, 17: 87-94 (2001)

建立siRNA knocked-downcellularsystems • 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。

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。

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- Chiparrays 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. (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

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 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

Cluster analysis • Materials and methods 112 gene 使用 the EXPANDER package 去做 average-linkage hierarchical clustering

GO functional gene annotations • Materials and methods The gene ontology (GO) annotations Computational promoteranalysis • Materials and methods PRIMA software

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

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

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

生科 4 甲 蔡德峰