臺灣博碩士論文加值系統

藉由在人類胚胎幹細胞做shRNA高通量篩選，我發現了一個新穎的多潛能調控基因ATF1。在降低ATF1在人類胚胎幹細胞的表現量時，發現神經外胚層的標誌基因會增加，但內胚層、中胚層及滋養外胚層的標誌基因卻沒有改變。值得注意的是，無論是在維持自我更新或是促進神經外胚層分化的培養環境中使用短髮夹RNA (shRNA)、干擾RNA（siRNA）去減少ATF1在人類胚胎幹細胞的表現量或是利用CRISPR/Cas9基因編輯技術剃除ATF1基因都足以使SOX2和PAX6表現量上升。此外，若是在神經外胚層誘導的培養環境中過量表現ATF1則會壓制神經外胚層分化的程度。而內生性的ATF1表現量在外胚層誘導時，會在1-3天後以後自發地減少。藉由雙重抑制ATF1與SOX2表現量發現PAX6和SOX1表現量則會受到抑制。顯示ATF1經由SOX2來調控PAX6 和SOX1表現量。利用螢光素酶檢測系统則確認了ATF1能在SOX2基因表現的調控上作為一種抑制表現的調控因子。總結以上的實驗與發現，我發現了ATF1的新穎作用，而這些研究結果相信對人類胚胎幹細胞在極早期的神經外胚層分化的調控上能有更進一步的理解。

By a shRNA high-throughput screen, I reveal that Activating Transcription Factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly upregulated neuroectoderm genes but not mesoderm, endoderm, and trophectoderm genes. Of note, downregulation or knockout of ATF1 with shRNA, siRNA, or CRISPR/Cas9 was sufficient to upregulate SOX2 and PAX6 expression under the undifferentiated or differentiated conditions, while overexpression of ATF1 suppressed neuroectoderm differentiation. Endogenous ATF1 was spontaneously downregulated after day(s) 1-3 of neural induction. By double knockdown experiments, upregulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shATF1 hESCs. Using the luciferase reporter assay, ATF1 acted as a negative transcriptional regulator of SOX2 gene expression. In summary, the novel function of ATF1 was discovered and these findings contribute to a broader understanding of the very first steps of regulating neuroectoderm differentiation in hESCs.

Table of Contents
致謝……………………………………………………………………………….....i
Table of Contents…………………………………………………………….…..…ii
List of Figures………………………………………………………………….……vi
List of Tables..............................viii
List of Appendices……………………………………………………………………..ix
中文摘要…………………………………………………………………….……….x
Abstract………………………………………………...……………………………xi
Chapter 1. Introduction………………………………...………………………….1
1-1 Embryonic stem cells and induced pluripotent stem cells………………………..1
1-2 The pluripotent regulatory circuit in ESCs ………………………….………...…2
1-3 Gene expression signature analysis in pluripotent stem cells……………..........3
1-4 High-throughput functional assays in hESCs………………………………........5
1-5 The application of ESCs/iPSCs derived neuron cells……………………………6
1-6 The novel functions of ATF1 in hESCs………….……………………..…..........7
Chapter 2. Materials and Methods………………………..………………………9
2-1. Cell lines and culture conditions…..…………………………………9
2-2. A shRNA high-throughput screen………...………………………………..10
2-3. Plasmids ……………………………………………………………………….11
2-4. Lentivirus production and hESC infection………………………12
2-5. RNA extraction and quantitative Real-Time PCR (qRT-PCR)….…..12
2-6. Western blot analysis ……………………………………………………..13
2-7. Flow cytometry for cell cycle analysis …………….……...14
2-8. Immunofluorescence assays ………………………………………………….14
2-9. Knockout ATF1 by CRISPR/Cas9 technique ……………………….15
2-10. Differentiation of shRFP/shATF1-expressing hESCs into neurons................16
2-11. Electrophysiology studies …...……………………………………………….17
2-12. Mesoderm and endoderm differentiation …………………….18
2-13. Primitive streak-like cell differentiation……………..18
2-14.Chromatin immunoprecipitation (ChIP).…………………...….19
2-15.Luciferase reporter assays…………………………………………………….20
2-16.Statistical analysis.………………………………….……………………..21
Chapter 3. Results………………………………………………………..……….22
3-1. Identification of pluripotency-related genes by RNAi screening in hESCs.....22
3-2. Knockdown of ATF1 decreases alkaline phosphatase activity but not AB activity in hESCs…………………………………….24
3-3. Downregulation of ATF1 specifically induces hESCs toward neuroectoderm differentiation ……………………………….25
3-4. Excludes an off-target effect of shATF1 by siRNA, a rescue experiment, and CRISPR/Cas9 knockout assay……………26
3-5. A decrease in ATF1 accelerates the expression of hESC neuroectoderm markers SOX2, PAX6, and SOX1 in a neural differentiation condition…………………….27
3-6. ATF1-downregulated cells can differentiate into functional neurons …….28
3-7. The ATF1 expression level is downregulated upon entry into the early stage of neuroectoderm differentiation but maintained during differentiation into mesoderm, endoderm, and primitive streak cells ………29
3-8. Overexpression of ATF1 suppresses neuroectoderm differentiation of hESCs in neural differentiation condition ..……………………………………………..……..30
3-9. ATF1 binds to the SOX2 promoter and negatively regulates SOX2 promoter activity....31
3-10. ATF1 regulates the expression of PAX6 and SOX1 through SOX2...………...32
Chapter 4. Discussion………………………………………………………….……33
References……………………………………………………………………..…….39
Figures………………………………………………………………….………………..52
Tables…………………………………………………………………………..………76
Appendix ………………………………………………………………………………..82

List of Figures
Fig. 1. Strategy to identify critical regulators for hESC self-renewal by a functional shRNA screen ….…………….……..……...52
Fig. 2. Downregulation of ATF1 decreases the expression level of the hESC undifferentiated marker alkaline phosphatase (ALP) but not cell number (Almarblu, AB)………………………………………………..54
Fig. 3. Downregulation of ATF1 induces hESC neuroectoderm differentiation in undifferentiation medium …………......56
Fig. 4. To exclude the off-target effect of shATF1, SMARTpool si-ATF1, a rescue experiment, and CRISPR/Cas9 experiments were used to examine the expression of neuroectoderm genes……………58
Fig. 5. Downregulation of ATF1 upregulates the expression levels of neuroectoderm markers in shATF1 expressing HUES-6 cells ………60
Fig. 6. The decrease in ATF1 upregulates the expression of hESC neuroectoderm markers SOX2, PAX6, and SOX1 in neural differentiation condition ……...….62
Fig. 7. ATF1-downregulated cells can differentiate into functional neurons ………….…64
Fig.8. The expression levels of ATF1 are decreased during neural induction and maintained while hESCs differentiate into mesoderm, endoderm, or primitive streak…...........66
Fig. 9. Overexpression of ATF1 suppresses neuroectoderm differentiation…...…………68
Fig.10.Downregulation of ATF1 increases the expression levels of the hESC neuroectoderm markers SOX2, PAX6, and SOX1 in undifferentiation condition (Day1-3)…………………………………………………………………………………………70
Fig.11. ATF1 binds to the SOX2 promoter and acts as a negative regulator of SOX2 expression ……………………………………………………...72
Fig. 12. Downregulation of ATF1 and SOX2 decreases neuroectoderm gene expression..................74


List of Tables
Table 1. List of microarrays from different cell types or tissues........76
Table 2. Differentiated express gene list………………………….………..…77
Table 3. Lethal gene list ………………….......………..……………………………….78
Table 4. ALP downregulated list………………………………………………………79
Table 5. AB downregulated list…………………………………………………………80
Table 6. List of primer set used in real-time PCR………………………81

List of Appendices
Appendix1: Antibody for Western blot…………………………..……..……………..82
Appendix2: Antibody for Immunofluorescence assay ……………………..83
Appendix3: Table 1 References…………………………..……..……………….……..84

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