臺灣博碩士論文加值系統

核仁磷酸蛋白 (NPM,B23) 是一個位於核仁的多功能蛋白，參與在核糖體生成、細胞凋亡、分化與細胞週期進行時的調控。近年來，B23被發現與許多轉錄因子結合而共同調節影響下游基因的表現，但是對於B23參與在基因表現的機制與角色仍有待研究。在此，我們利用整合性基因體技術 Microarray 和 ChIP-on-chip 來廣泛搜尋可能受到B23調控的基因，並更進一步利用實驗來確認基因的表現。利用此整合方法，我們成功找到了八個基因表現可能受到B23 的調控，分別是基因H2AC, H2BK, H2BF, H3G, CFB, NGEF, GNB1和KIF5B，當中針對histone 基因做更深入的研究探討。研究發現在基因上游啟動子處，當B23受到抑制時，屬於histone的活化因子 NPAT 的表現和 histone H4 的乙醯化程度皆增加，代表著此時histone 啟動子活化進而使基因表現增加。在細胞週期進入S phase時，B23 表現下降會增加且延長histone 基因表現，而細胞週期的進行也會受到延遲。代表B23是作為抑制因子來調控 histone 的啟動子並影響到基因的表現並參與細胞週期的調控。而在細胞分化過程中，我們發現B23 也參與在抑制 histone 啟動子的活性，進而使基因表現下降。因此本篇研究中發現到一個新的 histone 抑制因子 – B23，參與調控histone 的轉錄過程。未來將更進一步研究受到B23 抑制的確切機制與探討在生物體上所代表的意義

Nucleophosmin (NPM, B23) is a multifunctional protein that plays crucial role in ribosome biogenesis, cell cycle progression, apoptosis and cell differentiation regulation. In recent years, B23 has been found to interact with many transcription factors to regulate gene transcription. However, the exact role of B23 in the transcription regulation is still unclear. Here, we undertook an integrated functional genomic approach, which combines microarray and ChIP-on-chip analyses, to globally identify putative B23-target genes. Real-time PCR and chromatin precipitation (ChIP) assay were used to further confirm the putative B23-target genes. Through such integration approach, we have so far identified several candidate genes of B23-controlled transcription and chosen one of the candidates - histone genes for further characterization. ChIP assay revealed B23 occupancy of several histone gene promoters and that two markers of histone gene activation, NPAT and histone H4 acetylation, were upregulated in the absence of B23. During S phase, siRNA-mediated knockdown of B23 prolonged the expression of histone genes, transcription of which is normally restricted to early S phase. Therefore B23 may act as a repressor for histone genes and also a regulator of S phase progression. Our findings further indicated that B23 may participate in negatively regulating histone genes expression during cell growth arrest and differentiation. In the future we will focus on elucidating the molecular mechanism underlying B23-mediated regulation of histone genes and investigating its biological relevance.

Table of Contents

Introduction ...……………………………………………….………......1
Materials and Methods …………...…………….……………………... 4
Cell culture ………………………………………………………. 4
Microarray analysis ……………………………………………….4
ChIP-on-chip analysis …………………………………………….4
Cell transfection ………………………………………...………...4
RNA extraction and reverse transcriptase PCR …………………..5
Real-time PCR ……………………………………………………5
Cell cycle synchronization ………………………………………..5
Chromatin immunoprecipitation .…………………………………6
Immunobloting ……………………………………………………7
Oligonucleotide pull-down assay …………………………………7
Silver staining ……………………………………………………..8
Results …………………………………………………………………9
Identification and analysis of the candidate of B23-controlled genes
……………………………..………………………………………9
B23-mediated change of gene expression profile ……….....9
The occupancy of B23 on gene promoter regions ………….9
The link between B23 promoter binding and gene transcription regulation ……………………………………10
Biological role of B23 in histone genes expression regulation ….11
Effect of B23 on histone promoter regions ……………….11
Cell cycle-dependent regulation of histone gene expression ………………………………………….……11
B23-regulated histone gene expression during cell differentiation ……………………………………………..12
The underlying mechanism of B23-mediated histone gene repression ………………………………………………….14
Discussion …………………………………………………………….. 16
References …………………………………………………………… 19
Appendix ……………………………………………………………. 55


Table of Figures and Tables

Figure 1. RNAi-mediated knockdown of B23 expression in HeLa cells..23
Figure 2. Gene ontology analysis of positive genes selected from the microarray analysis ……………………………………………………..24
Figure 3. The integration of ChIP-on-chip and cDNA microarray analyses for identifying B23-targeted genes ……………………………………..28
Figure 4. The confirmation of putative B23-target gene expression …...33
Figure 5. The occupancy of B23 on gene promoter regions ……………35
Figure 6. Downregulation of B23 affects both NPAT binding and histone acetylation on histone promoter regions ………………………………..37
Figure 7. Downregulation of B23 leads to increased histone protein expression ……………………………………………………………… 38
Figure 8. Decreased cell growth rate in HeLa siB23 stable clones …….39
Figure 9. RNAi-mediated down-regulation of B23 delays S phase progression ……………………………………………………………...40
Figure 10. Expression of selected histone pair genes during S phase ….41
Figure 11. Regulation of histone promoters during S phase progression.44
Figure 12. Confirmation of histone H1 and H4 expression during cell differentiation ………………………………………………...…………47
Figure 13. Induced S phase arrest after extending culture in K562 cell...48
Figure 14. Extending culture triggered K562 cell differentiation……….49
Figure 15. Expression of other histone genes in K562………………….50
Figure 16. B23 is linked to the degree of histone H4 acetylation on histone promoter regions………………………………………………..51
Figure 17. Oligonucleotide pull-down assay reveals B23 occupancy on histone promoter regions ………………………………………………..52
Figure 18. Identification of putative histone promoter-associated proteins………………………………………………………………….53
Figure 19: The alteration of associated-proteins profile………………...54
Table 1. cDNA microarray analysis of gene expression in HeLa cells....27
Table 2. Putative B23-target genes ……………………………………..36
Table 3. Cell differentiation-dependent histone H1 and H4 expression is under the control of B23 ………………………………………………..46

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