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首页> 外文期刊>Nucleic Acids Research >Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq
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Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

机译：生物色谱动力学：通过校准ChIP-seq测量整个基因组中蛋白质占有率的通用方法

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Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.
机译：在体内与甲醛（称为ChIP-seq）进行交联后，与蛋白质相关的DNA片段测序已广泛用于描述蛋白质在基因组中的分布。未被广泛理解的是，该方法仅估计蛋白质的分布并且不能揭示样品之间的占有率变化。为此，我们在酿酒酵母和光滑念珠菌中用相同的表位直系同源蛋白标记，它们的序列差异很大，以至于大多数长于50 bp的DNA片段仅是一种物种所独有。在染色质片段化和免疫沉淀之前，通过将一定数量的光滑毛孢菌细胞（校准基因组）与啤酒酵母样品（实验基因组）混合，可以得出定量的占用率（占用率-OR），从而实现不仅在基因组内部而且在基因组之间的占有率的比较。我们首次证明了这种“内标”校准方法满足了量化ChIP-seq曲线的正弦条件，即在宽范围内的线性度。至关重要的是，通过使用功能性标记蛋白，我们的校准过程描述了一种将ChIP-seq谱图与背景噪声区分开来的真正方法。我们的方法不仅适用于高度保守的蛋白质，还适用于任何蛋白质，并且无需使用qPCR进行耗时，昂贵且技术要求高的ChIP定量分析，而qPCR只能在单个基因座上进行。正如我们在本文中首次证明的那样，经过校准的ChIP-seq代表了迈向记录蛋白质在不同细胞状态下沿染色体的定量分布的重要步骤，我们称其为生物色动力学。

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