Soragni Elisabetta, Kassavetis George A
Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634, USA.
J Biol Chem. 2008 Sep 26;283(39):26568-76. doi: 10.1074/jbc.M803769200. Epub 2008 Jul 30.
A major limitation of chromatin immunoprecipitation lies in the challenge of measuring the immunoprecipitation effectiveness of different proteins and antibodies and the resultant inability to compare the occupancies of different DNA-binding proteins. Here we present the implementation of a quantitative chromatin immunoprecipitation assay in the RNA polymerase III (pol III) system that allowed us to measure the absolute in vivo occupancy of pol III and its two transcription factors, TFIIIC and TFIIIB, on a subset of pol III genes. The crucial point of our analysis was devising a method that allows the accurate determination of the immunoprecipitation efficiency for each protein. We achieved this by spiking every immunoprecipitation reaction with the formaldehyde cross-linked in vitro counterparts of TFIIIB-, TFIIIC-, and pol III-DNA complexes, measuring the in vitro occupancies of the corresponding factors on a DNA probe and determining probe recovery by quantitative PCR. Analysis of nine pol III-transcribed genes with diverse sequence characteristics showed a very high occupancy by TFIIIB and pol III (pol III occupancy being generally approximately 70% of TFIIIB occupancy) and a TFIIIC occupancy that ranged between approximately 5 and 25%. Current data suggest that TFIIIC is released during transcription in vitro, and it has been proposed that TFIIIB suffices for pol III recruitment in vivo. Our findings point to the transient nature of the TFIIIC-DNA interaction in vivo, with no significant counter-correlation between pol III and TFIIIC occupancy and instead to a dependence of TFIIIB-DNA and TFIIIC-DNA complex maintenance in vivo on pol III function.
染色质免疫沉淀的一个主要局限性在于难以测量不同蛋白质和抗体的免疫沉淀效果，因而无法比较不同DNA结合蛋白的占有率。在此，我们展示了一种在RNA聚合酶III（pol III）系统中进行定量染色质免疫沉淀分析的方法，该方法使我们能够测量pol III及其两个转录因子TFIIIC和TFIIIB在一部分pol III基因上的绝对体内占有率。我们分析的关键在于设计一种能够准确测定每种蛋白质免疫沉淀效率的方法。我们通过在每个免疫沉淀反应中加入经甲醛交联的TFIIIB、TFIIIC和pol III-DNA复合物的体外对应物，测量相应因子在DNA探针上的体外占有率，并通过定量PCR确定探针回收率，从而实现了这一点。对九个具有不同序列特征的pol III转录基因的分析表明，TFIIIB和pol III的占有率非常高（pol III的占有率通常约为TFIIIB占有率的70%），而TFIIIC的占有率在约5%至25%之间。目前的数据表明，TFIIIC在体外转录过程中会释放，并且有人提出TFIIIB足以在体内招募pol III。我们的研究结果表明，TFIIIC与DNA在体内的相互作用具有瞬时性，pol III和TFIIIC的占有率之间没有明显的负相关，相反，TFIIIB-DNA和TFIIIC-DNA复合物在体内的维持依赖于pol III的功能。
