美国宾夕法尼亚州立大学开发并且证实一种新技术能够对解读和调控染色体---细胞内携带基因的绳状结构---的蛋白绘制图谱。染色体是由很多核小体串联而形成的，这些基因调节蛋白结合到核小体上的特异性次序决定着脑细胞、肝细胞或癌细胞是否形成。人们以前要精确确定这些蛋白结合到染色体上何处以及它们如何发挥作用是极其困难的。如今，他们利用这种新技术能够准确地找到了它们的结合位置，这样不论是它们正确地还是错误地调控整个基因组，人们都有可能拍出高分辨率的蛋白图谱。
2012年1月18日，这项研究提前在线发表在《自然》期刊上。他们最近也已将相关的研究发表在《细胞》期刊上。
Willaman分子生物学教授B. Franklin Pugh和研究生Ho Sung Rhee是利用称作核酸外切酶(exonuclease)的分子工具切除没有被其中一种基因调节蛋白结合的DNA序列来开始这一研究过程的。他们然后测定了每个仍然保持蛋白结合的DNA束(DNA bundle)的核苷酸序列，即DNA的四种主要碱基(用单字母表示为A，T，C和G)组成的序列。Pugh说，“这种被称作ChIP-exo的技术相对于其他技术的优势在于它能够将基因组上基因调节蛋白的结合位点从上百万到几十亿个核苷酸范围准确地缩小到大约1个核苷酸。这种改善就好比是从一台低分辨率的240p电视转变到一套高分辨率的1080p家庭影院系统。因此它能够为这些蛋白如何调控基因提供史无前例的高分辨率图谱。”
ChIP-exo技术也能消除检测系统产生的大量噪音，而这种问题对其他方法而言一直是个难题。他们利用这种更低噪音技术在染色体上发现了2到5倍多的基因调控蛋白的结合位点，从而为一种特定蛋白调控哪些基因以及更广泛地理解这些基因调节蛋白在整个基因组上的结构组装提供一幅更加完整的图谱。有了这种更加完整的图谱将允许科学家更加详细地理解人正常发育过程中基因途径如何发挥作用，抑或在疾病中它们如何失灵。(生物谷：towersimper编译)
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.
