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Investigating the Roles of Nap1, Taz2, and H1 on Nucleosome Assembly
Author(s) -
Blackwell Virginia Katherine,
Sarkar Prithwijit,
D'Arcy Sheena
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.777.3
Subject(s) - nucleosome , histone , linker dna , dna , biophysics , chemistry , biology , microbiology and biotechnology , biochemistry
In eukaryotic cells, DNA is packaged into the nucleus by extensive coiling, the most fundamental unit of which is the nucleosome. Nucleosomes are comprised of 146 base pairs of DNA wrapped around an octameric histone core, along with linker histone H1, controlling access to the genome. Proteins such as nucleosome assembly proteins (Naps) and p300 interact to shuttle histones to and from the nucleosome, regulating gene expression. In this project, we investigated the molecular details of interactions between Nap1 and the Taz2 domain of p300, their interactions with H1, and studied the role of these protein complexes on nucleosome dynamics. We purified Nap1, Taz2, and H1 using column chromatography, then assayed interactions between Nap1‐Taz2 and Nap1‐H1. We also performed experiments investigating the formation of a ternary complex of Nap1‐Taz2‐H1. Electrophoretic mobility shift assay (EMSA) and size exclusion chromatography with multi‐angle light scattering (SEC‐MALS) were used to study binding and determine the stoichiometry of these protein‐protein interactions. With these methods, we observed that Nap1 and Taz2 interact in vitro at an equimolar stoichiometry. We performed nucleosome assembly/disassembly assays in the presence of these protein complexes to study their effects on the nucleosome. Since Nap1, p300, and histones are a few of many ‘gatekeepers’ to our genetic code, it is critical that we determine how they function and cooperate to protect DNA. It is known that both Nap1 as well as p300 mutations contribute to cancer. While there is still much to unravel, p300 and interacting proteins are crucial to proper cell growth and division. This study furthers our understanding of nucleosome dynamics. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .