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.