臺灣博碩士論文加值系統

近幾年的研究發現在真核生物中，去氧核醣核酸的甲基化跟染色質蛋白的修飾是調控基因表現的最主要兩種附基因體調控機制。在這篇論文中首先我先用bisulfite 定序跟染色質體沉澱法證實了小鼠的p53，c-myc，c-fos和r-ras這些有表現的基因在基因體結構組成上分成兩個區域:區域一是去氧核醣核酸沒有甲基化和乙醯化的染色質蛋白和附著低度的染色質蛋白H2B/2A所構成的對核酸酶敏感的區域，而區域二是甲基化去氧核醣核酸和無乙醯化的染色質蛋白和附著高度的染色質蛋白H2B/2A所構成的對核酸酶不敏感的區域。其次，我又利用染色質體沉澱法發現CTCF，PARP 1，topoisomerase，Brg 1，HDAC 1和Dnmt 1這些附基因體調控因子能夠附著在這些有表現基因的啟動子區域和在這些基因的附基因體結構上的這兩個區域之間的邊界區域中。在我的研究中利用染色質沉澱法和染色體結構補獲法也發現在有絲分裂的細胞中會因為染色質蛋白H1附著在區域二形成高等結構而導致進行的RNA 鏈鎖聚合酶在這個區域中停留，而相反地在區域一中還保留其原本結構且RNA 鏈鎖聚合酶在此區域也不會受到影響進行轉錄的進行。另外，我加入PARP 1，topoisermase和HDAC 1的抑制劑到PT 67細胞中發現到這些基因的區域二結構也能夠影響RNA 鏈鎖聚合酶的進行。總合這些實驗的結果我得到一個新的理論就是這些調控附基因體因子會附著在有表現的基因上而形成兩個附基因體結構區域，而其中區域二會調控RNA 鏈鎖聚合酶的進行，區域一它則提供一個調控基因的記億體機制系統，能夠使得調控這些基因的轉錄因子跟RNA 鏈鎖聚合酶在細胞週期中不必重新運轉跟附著。

DNA methylation and histone modifications constitute the major epigenetic control mechanism of gene expression in eukaryotic cells. In this thesis, I first analyzed the organization of epigenetic domains of mouse p53 and four active genes by bisulfite sequences and ChIP techniques and showed that these genes were organized into two major epigenetic chromatin domains with differential histone modifications, DNA methylation and nuclease sensitivity. Second, I found that CTCF, PARP 1, topoisomerases, HDAC 1 and Dnmt 1 were associated with the promoter and the intron near the boundary of epigenetic domains. In mitotic cells, RNA polymerase II is depleted in domain II of p53 gene which is folded into higher order structure and associated with H1 histone whereas domain I conformation as preserved. Similar results were obtained in other four active genes in mitotic cells. Inhibition of PARP 1, HDAC 1, Topo II and Topo I also caused the premature transcription termination. The DNA sequence of intronic region of p53 gene containing CTCF binding site was found to exhibit strong enhancer activity. These results suggest the p53 and active genes are organized into novel epigenetic domains and the regulatory factors bound at the domain boundary are involved in the regulation of transcription elongation into the methylated chromatin domain. These results also suggest that depletion of RNA polymerase II is the result of physical barrier due to folding of chromatin in domain II. The novel chromatin structure in the first domain during mitosis also
suggests a mechanism for marking active genes in successive cell cycles.

摘要-------------------------------------------------------------------------------1
Abstract--------------------------------------------------------------------------2
Introduction---------------------------------------------------------------------3
DNA methylation-------------------------------------------------------------------------------4
Histone modification---------------------------------------------------------------------------6
The interplay between DNA methylation and histone modification in gene transcriptional regulation-----------------------------------------------------------------------8
A role for Insulators in nuclear organization for gene regulation------------------------10
Aim-----------------------------------------------------------------------------------------------12
Methods and Materials-----------------------------------------------------------------13
Results-----------------------------------------------------------------------------------------23
p53 gene contains two epigenetic domains with differential DNA methylation characteristic------------------------------------------------------------------------------------23
c-myc, c-fos, r-ras and ran also contain two DNA methylation epigenetic domains whereas silenced N-myc and Lmyc1 genes are fully methylated-------------------------24
Differential histone modification patterns associated with DNA methylation domains------------------------------------------------------------------------------------------25
Unmethylated epigenetic domain I in p53 gene is hypersensitive to nuclease and deficient in H2a/H2b---------------------------------------------------------------------------27
Unmethylated epigenetic domain I in active genes is hypersensitive to nuclease digestion-----------------------------------------------------------------------------------------28
Association of CTCF with both promoter and intragenic region of p53---------------29
PARP 1 colocalized with CTCF binding region-------------------------------------------30
Brg1 localized at boundary region in p53 gene--------------------------------------------31
Dnmt1 and HDAC1 colocalization in the p53 gene---------------------------------------31
Association of topoisomerase with the boundary region of p53 gene------------------32
Association of chromatin modification factors with epigenetic domains of active genes but not with inactive genes ------------------------------------------------------------32
Depletion of RNA polymerase II in epigenetic domain II of p53 gene in mitotic arrested cells with loss of regulatory factors from the methylated epigenetic domain-------------------------------------------------------------------------------------------33
Depletion of RNA polymerase II in epigenetic domain II of c-myc gene in mitotic arrested cells with loss of regulatory factors from the methylated epigenetic domain-------------------------------------------------------------------------------------------35
Inhibition of PARP1 resulted in the depletion of RNA polymerase in the epigenetic domain II and loss of regulatory factors----------------------------------------------------36
Inhibition of topoisomerases resulted in the depletion of RNA polymerase in the epigenetic domain II and loss of regulatory factors---------------------------------------36
Opposite effect of trichostatin A on transcription of p53 and c-myc versus c-fos and r-ras----------------------------------------------------------------------------------------------37
Association of histone H1 with epigenetic domain II of p53 chromatin during blocking of transcription elongation---------------------------------------------------------38
Change of higher order chromatin structure in epigenetic domain II and preservation of extended conformation and active histone modification codes of epigenetic domain I in mitotic cells and in cells treated with inhibitors---------------------------------------39
Enhancer activity of sequence at the intronic regulatory factor binding region--------40
Discussions-----------------------------------------------------------------------------------42
Figures-----------------------------------------------------------------------------------------50
Tables-------------------------------------------------------------------------------------------93
References-----------------------------------------------------------------------------------110

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