間白素結合因子(Interleukin enhancer binding factor, ILF)是從Hela 與 Jurkat cDNA 基因庫中所選殖出來的細胞轉錄因子，全長含有655個胺基酸。功能上，ILF藉著其DNA結合區與人類後天免疫不全病毒I型長端點重複部分(HIV-1 LTR)及間白素II型啟動子 (IL-2 promoter)的purine-rich motif 結合，進而活化HIV-1與IL-2基因表現。ILF的DNA結合區經序列比對與winged helix/forkhead (WH/FOX) 轉錄因子有35~89%的相似性，因而將ILF歸類為WH/FOX家族之蛋白。在WH/FOX家族中，其DNA結合區由兩個翅狀結構 (W1、W2)，三個α-helix (H1、H2、H3)及三個β-strand (S1、S2、S3)，以H1-S1-H2-H3-S2-W1-S3-W2方式組成。主要利用Helix 3與DNA的major groove作用。然而不同於WH/FOX家族之其他蛋白，利用NMR所決定ILF的DNA結合區之結構，在C端W2區域多出Helix 4的結構，此結構是否具有特殊功能目前尚不清楚。這Helix 4的結構反而與另一族ETS轉錄因子具有類似性，此ETS家族與WH/FOX家族同屬於winged helix-turn-helix (wHTH)家族。Ets-1的DNA結合區稱為ETS domain，而ETS domain的N端外側HI-1、HI-2，會和H1和C端H4這些α-helix形成inhibitory module，構成緊密複合體而抑制DNA結合；Ets-1以此自我抑制(autoinhibition)機制來調節與DNA結合。另外，inhibitory module 的N端外側之4個serine被磷酸化則會加強此抑制效果。我們藉二級結構預測發現，由ILF的DNA結合區往N端延伸，找到可能形成2個α-helix結構的區域。因此我們推測ILF具有類似Ets-1的自我抑制機制，亦即利用C端Helix 4與N端側延伸部分的2個α-helix形成所謂inhibitory module，以此來調節與DNA結合。我們設計構築出不同長度片段的ILF : ILF101(251-351)、ILF131(221-351)、ILF141(211-351)與ILF153(202-354)，並且利用Electrophoretic Mobility Shift Assay (EMSA)研究不同片段ILF對不同DNA的結合能力。所選用的DNA包括不同長度的WRT (WRT是一段由random primer 篩選出與myocyte nuclrear factor結合的DNA序列) 以及CASTING probe。利用CCCGGCAAAACATCAA當DNA probe，與各不同長度片段的ILF蛋白進行結合，分析EMSA數據並計算Kd比較各片段ILF結合能力的差異。Kd大小依序 : ILF153， ILF101 > ILF131， ILF141；其Kd值範圍為90-420 nM，愈大Kd值表示結合力愈小，故ILF131的結合能力最小。同時發現具有core sequence : GTAAACA或CAAAACA序列的短片段DNA與ILF有專一且強烈的結合，另外，當我們把含core sequence的DNA刪除5’端nucleotide使長度縮短，則結合現象喪失。推測推測是源自於DNA的5’端長度不足，使Wing 2無法與DNA作用而不能穩定該結合複合體。我們推測ILF131對DNA結合能力的調節，可能利用類似Ets-1的自我抑制機制，接下來我們或許可以探討磷酸化對自我抑制的調控;至於具DBD往N端延伸30個胺基酸的區域是否形成所預期的α-helix結構，可能需要日後藉助NMR分析。

Interleukin enhancer binding factor (ILF) is a transcription factor which consists of 655 amino acids and binds to the IL-2 promoter. Although the function of ILF is not well understood, previous studies suggest that they might have a positive role in regulating IL-2 gene expression. The DNA-binding domain of ILF is a member of the winged helix/ forkhead family and consists of four α-helices, three β-strands, one type I turn, and one wing, arranged in the order H1-T1-S1-H2-H3-S2-W1-S3-H4. Interestingly, in contrast to other proteins of this family, the DNA-binding domain of ILF contains a C-terminal alpha-helix in place of a typical Wing 2. This extra alpha-helix is stabilized by the hydrophobic interactions between H4 and H1. This structural difference may be responsible for the different DNA-binding specificity of ILF compared to other winged helix /forkhead proteins. Ets-1, a member of winged helix transcription factors, also has the Helix 4 structure and has been shown to exhibit an intramolecular autoinhibition mechanism to regulate its binding to DNA. Additional two α-helices (HI-1 and HI-2) at the N-terminus of the DNA-binding domain of ETS-1 interact with H4 to form an inhibitory module to repress its DNA-binding affinity. In addition, the calcium-dependent phosphorylation represses the DNA binding of Ets-1 by reinforcing autoinhibition. Four serine residues mediated this inhibitory effect are located at the N-terminus of the DNA-binding domain. Based on secondary structure analysis, we found that the N terminal region of the DNA-binding domain of ILF may form two α-helices. Therefore, we propose that ILF may regulate the DNA binding activity by autoinhibition mechanism that was used by Ets-1. In this study we expressed four fragments of ILF101(251-351), ILF131(221-351), ILF141(211-351), and ILF153(202-354) in E. coli and study the relative binding affinity of four fragments of ILF with different DNA probes by electrophoretic mobility shift assay (EMSA). The EMSA study showed that the relative binding affinities of the four proteins with the probe CCCGGCAAAACATCAA were ILF153, ILF101 > ILF131, ILF141. The Kd values of the fragments of ILF and the probes are ranging from 90-420 nM. We also found that the probes with the sequences GTAAACA or ATAAACA can specifically bind to the DNA-binding domain of ILF. The study on the phosphorylation effect of the N-terminus of the DNA-binding domain of ILF on its DNA binding is ongoing. This study demonstrates a similar autoinhibition mechanism among the winged helix transcription factors.

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