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Functional and Structural Insights into RSC‐mediated Nucleosome Remodeling via In‐Vivo Crosslinking
Author(s) -
Evans Brian,
Wilkins Bryan
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.777.7
Subject(s) - chromatin structure remodeling (rsc) complex , nucleosome , chromatin remodeling , chromatin , histone , microbiology and biotechnology , histone code , histone octamer , histone h2a , biology , chemistry , histone modifying enzymes , histone h3 , dna , genetics , computational biology
In eukaryotes, genetic information is stored as chromatin. Within the chromatin, DNA is wound around octameric units of histone proteins. The histone‐DNA complex forms the nucleosome and represents the most basic level of DNA compaction. A class of proteins known as chromatin remodelers physically reposition nucleosomes along the chromatin fiber. This can directly affect and mediate many important processes such as DNA replication, repair, and transcription. Albeit, due to their large sizes and numerous auxiliary subunits, chromatin remodeler interactions at the nucleosome have yet to be fully characterized and remain relatively obscure. The RSC complex is the most abundant remodeling complex of budding yeast, with homologs in both animals and plants. In this work, we use synthetic biology to trap histone‐RSC remodeler interactions in living cells using the unnatural amino acid p‐benzoylphenylalanine (pBPA). We site‐specifically insert pBPA into histones and identify remodeler binding via the addition of short peptide fusion tags to our interaction target protein. This system has the potential to illuminate chromatin remodeler complex interactions which can then be used to identify and assign biologically important structures. By properly implementing the system, we exemplify the potential of this technology for creating crosslinking maps for RSC. This conclusion is supported by Western blot analysis and a clearly identified crosslink between a myc‐tagged subunit of RSC (Sth1 protein) with histone H2A and histone H3. In addition, we show that binding to H3 (but not H2A) is dependent upon PTMs and a defined acetylation event at lysine 14 on histone H3. In cells that lack H3 K14ac, Sth1 can no longer be sequestered to the nucleosome providing in vivo mechanistic details about RSC‐nucleosomal function/structure relationships. Support or Funding Information NIH R15 Academic Research Enhancement Award (AREA) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .