Fluorescence polarization reveals a possible displacement  model of competition in PRC2:RNA:DNA interactions

Fenske, Regan Lynn

Undergraduate Honors Thesis

Fluorescence polarization reveals a possible displacement model of competition in PRC2:RNA:DNA interactions Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/5x21tg835

Abstract

PRC2 is a histone methyltransferase that acts on histone subunit H3 at lysine 27 to repress chromatin state and inhibit gene expression. The interactions between PRC2 and RNA have been heavily studied in vivo and in vitro with conflicting results reported on the relationship, but there is less data on interactions between PRC2 and DNA. Fluorescence polarization-based methodology was used with various RNA and DNA species to study the binding kinetics of PRC2 with RNA and DNA. Previous data indicate that PRC2 has a relatively high affinity for DNA species that are rich in consecutive G and C nucleotides. Double-stranded DNA species with lengths of 50-60 bp were designed for this project. Fluorescence polarization binding experiments were used to identify the general binding affinity of the DNA and RNA species to PRC2 by calculating the Kd apparent for the binding curve. After the apparent binding affinities were determined, various FP-competition experiments were performed to determine if each DNA species could be competed off by an RNA species or itself, and vice versa. These results showed that the DNA species were more effective competitors although the RNA species were stronger binders. These results suggest a mechanism for RNA-mediated PRC2 regulation that could reconcile the conflicting experimental results and interpretations of past experiments. Further FP-based Kd experiments under varying salt concentrations revealed PRC2 has ionic interactions with DNA that are not seen with RNA. This suggests PRC2 has extra unique interactions with DNA, which could explain the differing effectiveness of DNA versus RNA as competitors. Altogether, the results imply that DNA has additional contacts with PRC2 that limit its displacement by RNA, allowing for RNA to guide PRC2 to its target genetic loci and then for PRC2 to deposit its methyl marks without being sequestered by RNA.

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