https://scholars.lib.ntu.edu.tw/handle/123456789/160330
標題: | dBRWD3 抑制 Polycomb Group 突變導致之異位基因表現 dBRWD3 suppressed the ectopic gene expression resulted from Polycomb Group mutations |
作者: | 陳怡君 Chen, Yi-Jyun |
關鍵字: | 表觀遺傳學;轉譯;Polycomb Groups;dBRWD3;組蛋白H3.3;異位基因表現;Epigenetics;Transcription;H3.3;Ectopic gene expression | 公開日期: | 2016 | 摘要: | 表觀遺傳調控為控制基因表現的一種方式,可透過改變染色質上核小體的位置、核小體中組蛋白的組成、核小體中組蛋白上的轉譯後修飾及去氧核醣核酸序列中腺嘌呤的甲基化,來達到染色質的狀態改變,影響轉錄因子接近染色質的能力而改變基因表現,進而影響生物體中的細胞型態、細胞分化或不同發展階段的基因調控。其中,可透過加上組蛋白的轉譯後修飾達到抑制基因表現的一種蛋白質複合體Polycomb Group (PcGs) 可專一性地催化H2AK118及H3K27me3此二種抑制性組蛋白密碼,來使其目標基因的表現被抑制。除此之外,PcGs也被發現可調控具有轉錄因子特性的基因、或是參與在訊息傳遞路徑中的部份基因表現。另一方面,核小體中組蛋白變異體的置換亦被視為一種表觀遺傳的調控機制,如組蛋白H3變異體H3.3在染色質上的置換與基因轉錄的區域相關。在實驗室先前的研究中發現,組蛋白變異體的置換存在負向的調控方式,意即H3.3的置換可被dBRWD3 (Drosophila Bromodomain and WD repeat domain containing 3) 透過其伴護蛋白HIRA/YEM的作用來負向調控。而在本研究中,我們透過減少PcGs其中一蛋白質Pc在果蠅幼蟲神經系統中的表現,發現其目標基因Ubx及Antp產生異位基因表現,而此異位基因表現可由減少dBRWD3的表現來達到抑制。而若減少PcGs中的H3K27甲基轉移酶E(z) 的表現,也會產生目標基因的異位表現,同樣地,亦可透過減少dBRWD3的表現來抑制。此外,當我們減少特定的基本轉錄因子 (General transcription factors, e.g. TAF5, TAF7, Cdk7 or CycH) 的表現時,同樣也抑制了由Pc或E(z) 表現量降低導致的異位基因表現。有趣的是,當我們只降低dBRWD3表現量時,Ubx或Antp的原位基因表現卻不受影響,因此,我們好奇降低dBRWD3表現量可達到抑制異位基因表現的分子機制為何。首先,我們著重於dBRWD3是否會對RNA Polymerase II產生影響做探討,利用DamID (DNA Adenine Methylation Identification) 來偵測Pol II在Ubx或Antp上佔據的頻率,發現dBRWD3的表現量降低可能會使Pol II停留在Antp上的啟動子或靠近5’端的區域,而影響基因轉錄初期。接下來,我們想知道dBRWD3表現量降低而抑制異位基因表現的原因,是否是透過補回因PcGs表現量降低導致的抑制性組蛋白密碼缺失,因此我們利用H2AK118ub或H3K27me3的染色質免疫沉澱 (ChIP),發現在降低Pc或E(z) 表現量的同時,再減少dBRWD3的表現時,抑制性組蛋白修飾並未受到影響,因此,其分子機制還有待更多實驗來釐清。而在了解dBRWD3在異位基因表現中扮演之角色的過程中,我們意外發現異位與原位基因表現在表觀遺傳中的不同之處。 Epigenetic regulation involves chromatin remodeling, nucleosome assembly, replacement of histone variants and DNA methylation. Histone modification alter the compaction of chromatin, thereby affecting transcription factors binding to gene regions. One of the well-known enzymes that catalyze histone modifications, Polycomb group (PcG) proteins, are reported to write the repressive histone marks, H3K27me3 and H2AK118ub, to keep the targeted gene silenced. On the other hand, the incorporation of histone variants is another regulatory mechanism of epigenetics. In the previous studies, we found that incorporation of histone variants could be regulated negatively. The incorporation of histone 3 variant, H3.3, could be negatively regulated by dBRWD3 (Drosophila Bromodomain and WD repeat domain containing 3) through a HIRA/YEM-dependent pathway. Here, we found when we depleted one of the PcG protein, Pc, in Drosophila larval nervous system, Ubx and Antp, two Hox genes, expressed ectopically (into the brain. Interestingly, this ectopic gene expression could be suppressed by dBRWD3 depletion. dBRWD3 depletion also suppressed the ectopic gene expression caused by the depletion of E(z), encoding the histone methyltransferase that catalyzes the modification of H3K27me3. Moreover, when we depleted the general transcription factors, Cdk7, CycH, TAF5 or TAF7, the ectopic expression of Ubx or Antp was also suppressed. Interestingly, there was no alteration of orthotopic gene expression by dBRWD3 depletion. To dissect the mechanisms underlying the suppression of ectopic gene expression by dBRWD3 depletion. First, we focused on the effect of dBRWD3 on RNA polymerase II. We utilized DamID (DNA adenine methyltransferase identification) to study the frequency of Pol II occupying on the target genes (Ubx, Antp). The results showed that dBRWD3 depletion might maintain Pol II-Dam on promoter and 5'' regions of Antp and then affect the initial stage of transcription.. Second, we wondered if the repressive histone mark added by PcGs would be rescued by dBRWD3 depletion. We utilized ChIP of H3K27me3 or H2AK118ub, and found that additional dBRWD3 depletion had no effect on these marks when E(z) or Pc was depleted. In the process to investigate the role of dBRWD3in ectopic gene expression, we unexpectedly provided a new insights into the differences between the ectopic and orthotopic gene expression. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/271753 | DOI: | 10.6342/NTU201603124 | Rights: | 論文公開時間: 2018/8/26 論文使用權限: 同意有償授權(權利金給回饋學校) |
顯示於: | 分子醫學研究所 |
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