美國賓夕法尼亞州立大學開發(fā)并且證實一種新技術(shù)能夠?qū)庾x和調(diào)控染色體---細胞內(nèi)攜帶基因的繩狀結(jié)構(gòu)---的蛋白繪制圖譜,。染色體是由很多核小體串聯(lián)而形成的，這些基因調(diào)節(jié)蛋白結(jié)合到核小體上的特異性次序決定著腦細胞,、肝細胞或癌細胞是否形成,。人們以前要精確確定這些蛋白結(jié)合到染色體上何處以及它們?nèi)绾伟l(fā)揮作用是極其困難的,。如今,，他們利用這種新技術(shù)能夠準確地找到了它們的結(jié)合位置，這樣不論是它們正確地還是錯誤地調(diào)控整個基因組,，人們都有可能拍出高分辨率的蛋白圖譜,。
2012年1月18日，這項研究提前在線發(fā)表在《自然》期刊上,。他們最近也已將相關(guān)的研究發(fā)表在《細胞》期刊上,。
Willaman分子生物學教授B. Franklin Pugh和研究生Ho Sung Rhee是利用稱作核酸外切酶(exonuclease)的分子工具切除沒有被其中一種基因調(diào)節(jié)蛋白結(jié)合的DNA序列來開始這一研究過程的。他們?nèi)缓鬁y定了每個仍然保持蛋白結(jié)合的DNA束(DNA bundle)的核苷酸序列,，即DNA的四種主要堿基(用單字母表示為A,，T，C和G)組成的序列,。Pugh說,，“這種被稱作ChIP-exo的技術(shù)相對于其他技術(shù)的優(yōu)勢在于它能夠?qū)⒒蚪M上基因調(diào)節(jié)蛋白的結(jié)合位點從上百萬到幾十億個核苷酸范圍準確地縮小到大約1個核苷酸。這種改善就好比是從一臺低分辨率的240p電視轉(zhuǎn)變到一套高分辨率的1080p家庭影院系統(tǒng),。因此它能夠為這些蛋白如何調(diào)控基因提供史無前例的高分辨率圖譜,。”
ChIP-exo技術(shù)也能消除檢測系統(tǒng)產(chǎn)生的大量噪音，而這種問題對其他方法而言一直是個難題,。他們利用這種更低噪音技術(shù)在染色體上發(fā)現(xiàn)了2到5倍多的基因調(diào)控蛋白的結(jié)合位點,，從而為一種特定蛋白調(diào)控哪些基因以及更廣泛地理解這些基因調(diào)節(jié)蛋白在整個基因組上的結(jié)構(gòu)組裝提供一幅更加完整的圖譜。有了這種更加完整的圖譜將允許科學家更加詳細地理解人正常發(fā)育過程中基因途徑如何發(fā)揮作用,，抑或在疾病中它們?nèi)绾问ъ`,。(生物谷：towersimper編譯)
doi:10.1016/j.cell.2011.11.013
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Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution
Ho Sung Rhee, B. Franklin Pugh
Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives (erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak pairs by deep sequencing. Contaminating DNA is degraded or fails to form complementary peak pairs. With the single bp accuracy provided by ChIP-exo, we show an unprecedented view into genome-wide binding of the yeast transcription factors Reb1, Gal4, Phd1, Rap1, and human CTCF. Each of these factors was chosen to address potential limitations of ChIP-exo. We found that binding sites become unambiguous and reveal diverse tendencies governing in vivo DNA-binding specificity that include sequence variants, functionally distinct motifs, motif clustering, secondary interactions, and combinatorial modules within a compound motif.
doi:10.1038/nature10799
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Genome-wide structure and organization of eukaryotic pre-initiation complexes
Ho Sung Rhee & B. Franklin Pugh
Transcription and regulation of genes originate from transcription pre-initiation complexes (PICs). Their structural and positional organization across eukaryotic genomes is unknown. Here we applied lambda exonuclease to chromatin immunoprecipitates (termed ChIP-exo) to examine the precise location of 6,045 PICs in Saccharomyces. PICs, including RNA polymerase II and protein complexes TFIIA, TFIIB, TFIID (or TBP), TFIIE, TFIIF, TFIIH and TFIIK were positioned within promoters and excluded from coding regions. Exonuclease patterns were in agreement with crystallographic models of the PIC, and were sufficiently precise to identify TATA-like elements at so-called TATA-less promoters. These PICs and their transcription start sites were positionally constrained at TFIID-engaged downstream +1 nucleosomes. At TATA-box-containing promoters, which are depleted of TFIID, a +1 nucleosome was positioned to be in competition with the PIC, which may allow greater latitude in start-site selection. Our genomic localization of messenger RNA and non-coding RNA PICs reveals that two PICs, in inverted orientation, may occupy the flanking borders of nucleosome-free regions. Their unambiguous detection may help distinguish bona fide genes from transcriptional noise.
