Department of Genetics, Texas Biomedical Research Institute , San Antonio, Texas 78227, United States.
J Proteome Res. 2017 Sep 1;16(9):3433-3442. doi: 10.1021/acs.jproteome.7b00448. Epub 2017 Aug 4.
The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.
着丝粒是动粒形成的染色体位置，对于确保细胞分裂过程中姐妹染色单体的正确分离至关重要。人们已经投入了大量的努力来理解着丝粒的特征和作用，但着丝粒的一些基本方面，如定义它的完整元素列表，仍然不清楚。众所周知，人类着丝粒包括高度重复的一类 DNA，称为α卫星或α 型 DNA。我们在这里首次对人类蛋白-α卫星相互作用进行了以 DNA 为中心的研究，采用了一种称为 HyCCAPP（染色质相关蛋白杂交捕获用于蛋白质组学）的方法，以鉴定人细胞系中α型卫星染色质的蛋白成分。使用 HyCCAPP，从细胞裂解物中分离交联的α卫星染色质，并通过质谱分析捕获的蛋白质。与对照下拉实验中鉴定的蛋白质进行比较后，有 90 种蛋白质被鉴定为在α型 DNA 上富集。该列表包括许多已知的着丝粒结合蛋白，以及多个新的α卫星结合蛋白，如 LRIF1，一种异染色质相关蛋白。HyCCAPP 能够揭示已知和新的α型 DNA 相互作用蛋白，突出了这种方法的有效性和实用性。
