Elucidating Protein–DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry

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（centromere）是染色体上动粒（kinetochore）形成的染色体位点，对于细胞分裂过程中姐妹染色单体的精准分离至关重要。尽管学界已投入大量精力探究着丝粒的特征与功能，但其定义的核心要素（如完整的元件列表）等基础科学问题仍未明确。众所周知，人类着丝粒包含一类高度重复的DNA序列，即α卫星（alpha satellite，又称类人猿卫星DNA）。本研究首次开展了人类蛋白质与α卫星DNA相互作用的以DNA为中心的分析，采用HyCCAPP（用于蛋白质组学的染色质结合蛋白杂交捕获技术，hybridization capture of chromatin-associated proteins for proteomics）方法，在人类细胞系中鉴定α卫星染色质的蛋白质组分。实验中，研究人员通过HyCCAPP从细胞裂解液中分离交联的α卫星染色质，并借助质谱（mass spectrometry）分析捕获得到的蛋白质。将其与对照下拉实验（pull-down）鉴定得到的蛋白质进行比对后，共鉴定出90种在α卫星DNA区域富集的蛋白质。该蛋白列表不仅包含诸多已知的着丝粒结合蛋白，还发现了多种全新的α卫星结合蛋白，例如异染色质相关蛋白LRIF1。HyCCAPP既能鉴定已知的、也能发现新的α卫星DNA互作蛋白，这一能力证实了该方法的有效性与应用价值。