AP-4是具有basic helix-loop-helix (bHLH) 功能區的轉錄調節因子(transcription factor)，並透過bHLH功能區與具特異序列 (5’-CAGCTG-3’) 的E-box DNA相互結合。目前許多證據指出，AP-4可調控許多細胞重要的生理功能，例如細胞生長、個體發育等。最近的研究顯示，AP-4會抑制與細胞生長及DNA損傷修補機制相關HDM2之轉錄，然而AP-4抑制HDM2轉錄的機制仍然未知。因此若能解開此轉錄調控的機制，將可讓吾人更加了解AP-4在細胞生長及DNA損傷修補機制中的生理功能。
本研究中，吾人首先發現了HDM2的P2 啟動子 (HDM2-P2 promoter) 上的E-box序列(5’-CAGCTG-3’)，並確認AP-4可透過與此E-box之結合，進而抑制HDM2之基因轉錄，吾人並進一步確認此抑制機制與p53無關。透過功能區缺失的研究，吾人發現AP-4上的glutamine/proline-rich功能區為抑制HDM2基因轉錄的重要功能區。綜合以上的實驗結果顯示，AP-4可能與未知的蛋白質結合，形成可以抑制HDM2基因轉錄的蛋白質複合體。
為了更進一步了解AP-4抑制HDM2轉錄的機制，吾人結合DNA 親和性純化及包含cICAT與iTRAQ標定方法的互補性定量蛋白體學方法，找出可與HDM2-P2上的E-box結合的AP-4蛋白質複合體。吾人在此AP-4蛋白質複合體75個蛋白質中，發現13個具有顯著性變化的DNA 修補機制相關的蛋白質，顯示AP-4可能直接或間接參與DNA損傷修復機制的運作。
此外，此AP-4蛋白質複合體亦包含轉錄調節因子、轉錄抑制因子、以及與轉錄抑制相關的組蛋白修飾酶，且AP-4可與其中數個蛋白質相互結合，如CTCF、SP1、以及HDAC1。當吾人進一步抑制細胞HDAC1酵素的活性時，發現並無法回復AP-4的轉錄抑制。此結果顯示AP-4並非透過以往已知與HDAC相關的轉錄抑制機制，達成抑制HDM2基因轉錄的作用。依據吾人之互補性定量蛋白體研究的數據，AP-4有可能透過在AP-4複合體中的祖蛋白甲基酶(histone methyltransferase)或合體重組蛋白複合體(nucleosome remodeling SWI/SNF complex)，經由組蛋白甲基化或核體重組的方式，進而抑制HDM2的基因轉錄。
總結來說，吾人藉由分子生物學方法，探討AP-4如何與HDM2之啟動子交互作用，達成抑制HDM2轉錄的目的。同時，吾人以單一性DNA親和性純化方式，初步分離與HDM2啟動子結合的AP-4蛋白複合體，並利用結合cICAT與iTRAQ標定方法的互補性定量蛋白體學方法，成功地分離出專一性的AP-4蛋白複合體。藉由分析此蛋白複合體的組成，吾人得以推論AP-4對於HDM2轉錄的可能抑制機制，並發現AP-4可能參與DNA損傷修補機制。此外，在本論文中吾人所提出的結合單一DNA親和性純化方式與互補性定量蛋白體學之方法，對於未來功能性蛋白質複合體之定量蛋白體學相關研究，提供了另一個有別於傳統生化純化方式的新策略。

Transcription factor activating enhancer binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism.
Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter in vitro and in vivo as demonstrated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Luciferase assay further revealed that AP-4 represses HDM2 transcription in a p53-independent manner. In addition, incremental truncations of AP-4 showed that the C-terminal glutamine/proline-rich domain is essential for transcriptional repression of HDM2.
To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we performed single-step DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex. The two labeling methods complementarily quantified 75 putative components in AP-4 protein complex, including the most significant recruitment of DNA damage–responsive proteins, followed by transcription factors, transcription repressors/corepressors, and histone-modifying proteins.
Using AP-4 truncation mutants and DNA pull-down assay, specific interaction of AP-4 with CTCF, SP1, and histone deacetylase 1 (an AP-4 corepressor) was validated. Although AP-4 may repress HDM2 transcripion by recruiting HDAC, inclusion of HDAC specific inhibitor, trichostatin A, did not alleviate AP-4-mediated repression of HDM2 transcription. Taken together the data suggest a previously unidentified histone deacetylase-independent repression mechanism. Alternatively, the complementary quantitative proteomics study suggests that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI/SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in a transcriptional regulating complex at the HDM2 promoter in response to DNA damage.
In conclusion, we successfully demonstrate how AP-4 regulates HDM2 transcription via binding to the previously unknown AP-4 binding site. By taking the advantage of the complementary quantitative proteomics, we identify a DNA bound AP-4 protein complex from single-step DNA afftinity purification from crude nuclear extracts. By analyzing the components and functions of the AP-4 protein complex, we are able to deduce the possible repressive mechanisms on HDM2 transcription and the potential role of AP-4 in DNA damaging response. Our findling may shed a light in better understanding the physiological role of AP-4. Finally, we also develop a strategy combining single-step purification and complementary quantitative proteomics for target proteomics study, which provides an alternative MS-based way to study protein complex in the future.

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