In Vivo Crosslinking and Mass Spectral Analysis of the Yeast Nucleosomal Protein Interactome.

Chromatin is essential for proper DNA storage and gene regulation. The repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around an octameric core of histone proteins. Histones interact with DNA, but they also make contacts to chromosomal regulating proteins. Histones undergo an array of dynamic posttranslational modifications (PTMs), each having an essential role in chromosomal structuring, function, and maintenance. To understand how nucleosomes regulate chromatin processes and how the dynamic PTM landscape influences histone-protein interactions, it is important to identify interacting partners. We use UV crosslinking unnatural amino acids that allow for site-selective photo-crosslinking from histones, in vivo. This allows for the study of chromatin protein-protein interactive dynamics in the physiological environment of the nucleus. We pair crosslinking techniques with mass spectral (MS) analysis to identify histone interacting proteins from cell lysates providing an unbiased overview of the site-specific interactome. Our assays reveal many well-established chromatin partners; however, they also provided insight to nucleosomal contacts that have been less reported in the literature. Of interest, we identify several histone contacts to ribosomal subunits, both large and small. We are interested in the extra ribosomal function of these proteins and their roles in chromatin regulation. To verify MS hits, we crosslink in yeast strains harboring a C-terminal fusion tag (Myc) to the gene of interest, while expressing the crosslinking histone with a separate tag (HA). Products are precipitated and analyzed via western blot by decorating with antibodies to the opposing tag. We have verified several histone-ribosomal protein interactions, including Rpl7A, Rpl3, and Rpl10. Using this technique, we investigate PTM mutations and their effects on the ability to form crosslinked products, providing insight on sequestering and regulatory mechanisms. These initial investigations suggest that UV crosslinking paired with MS analysis is an effective tool for studying yeast nucleosomal protein contacts. © FASEB.

Citation

Raven Fisher. In Vivo Crosslinking and Mass Spectral Analysis of the Yeast Nucleosomal Protein Interactome. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2022 May;36 Suppl 1

PMID: 35553455

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