Actions for Purification and Characterization of Chromatin Remodeler SNF2L

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Purification and Characterization of Chromatin Remodeler SNF2L

Author
Waldt, Hannah
Published
[University Park, Pennsylvania] : Pennsylvania State University, 2024.
Physical Description
1 electronic document
Additional Creators
Armache, Jean-Paul and Schreyer Honors College
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Restricted (PSU Only).
Summary
DNA is packed into chromatin to fit within the spatial constraints of the nucleus. The repeating unit of chromatin is the nucleosome, composed of histone octamers wrapped in DNA. The spacing of these nucleosomes and the accessibility of DNA have important implications in gene regulation. The extent of packing can be altered by enzymes called chromatin remodelers. These enzymes work by covalently modifying the nucleosome to relieve or enhance packing or by coupling ATP hydrolysis to nucleosome sliding. One of the enzymes belonging to the latter class is the ISWI protein, which works to center nucleosomes on DNA. This protein, initially discovered in Drosophila, has two human homologs, SNF2H and SNF2L. They are closely related, but the structural or functional difference between the two is not entirely known. Though SNF2H has been extensively characterized, the structure and function of SNF2L is much lesser understood. The aim of the research described in this thesis is to find optimal expression and purification approaches of SNF2L, which would allow us to characterize the structure of SNF2L and SNF2L bound to a nucleosome. A two-step purification scheme was developed and optimized to successfully isolate SNF2L in E. coli. Subsequently, the purified protein was mixed with nucleosomes and ran through a glycerol-gluteraldehyde gradient using an ultracentrifuge (a technique called GraFix). Negative stain images show that the remodeler is bound to the nucleosome forming a nucleosome + remodeler complex. Future studies will increase sample yield for use in cryo-EM and pursue the in-depth 3D characterization of different nucleotide states of the complex. Characterization of SNF2L and an inactive SNF2L variant may reveal key differences between the SNF2H and SNF2L homologs that allow for effective development of therapeutics.
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Dissertation Note
B.S. Pennsylvania State University 2024.
Technical Details
The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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