High-resolution mapping of transcription factor binding sites on native chromatin.
High-resolution mapping of transcription factor binding sites on native chromatin.
复制标题
DOI：
10.1038/nmeth.2766
复制
发表时间：
2014-02
期刊：
影响因子：
48
通讯作者：
Henikoff, Steven
中科院分区：
文献类型：
--
作者：
Kasinathan, Sivakanthan;Orsi, Guillermo A.;Zentner, Gabriel E.;Ahmad, Kami;Henikoff, Steven
序列特异的DNA结合蛋白，包括转录因子，是基因调控和染色质结构的关键决定因素。甲醛交联-超声化-染色质免疫沉淀(X-CHIP)是目前应用最广泛的TF结合谱分析方法，但分辨率低、特异性和敏感性差。我们提出了一种简单的方法，从微球菌核酸酶消化未交联染色质开始，然后亲和纯化TF和配对末端测序。酿酒酵母Abf1和Reb1的有机(来自亲和纯化的自然分离染色质的基因组占据区域)图谱提供了高度准确的碱基对分辨图谱，不偏向可接近的染色质，也不需要输入标准化。我们还通过对果蝇、黑腹果蝇、Gaga因子和PinSqueak的分析，证明了我们的方法在应用于更大的基因组时具有高度的特异性。我们的结果表明，有机图谱是一种广泛适用的高分辨率方法，可以灵敏和特异地分析蛋白质与DNA的直接相互作用。
Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.
登录
查看更多内容
影响因子：
5.3
作者：
Gelbart, ME;Rechsteiner, T;Tsukiyama, T
通讯作者：
Tsukiyama, T
影响因子：
5.3
作者：
LANG, WH;REEDER, RH
通讯作者：
REEDER, RH
影响因子：
64.5
作者：
Hartley PD;Madhani HD
通讯作者：
Madhani HD
影响因子：
3.5
作者：
Beinoraviciute-Kellner, R;Lipps, G;Krauss, G
通讯作者：
Krauss, G
影响因子：
48
作者：
Hesselberth, Jay R.;Chen, Xiaoyu;Zhang, Zhihong;Sabo, Peter J.;Sandstrom, Richard;Reynolds, Alex P.;Thurman, Robert E.;Neph, Shane;Kuehn, Michael S.;Noble, William S.;Fields, Stanley;Stamatoyannopoulos, John A.
通讯作者：
Stamatoyannopoulos, John A.