Is the Oligomerization Domain of GOF mtp53 R273H Critical for Chromatin-Based Activities?
Annor1,2, C. Freedman2, V. Elison2, G. Xiao2, J. Bargonetti1,2,3*
1The Graduate Center- City University of New York (CUNY). 2The Department of Biological Sciences Hunter College, 3Weill Cornell Medical College, New York City
In triple negative breast cancers (TNBC), there is a high occurrence of missense mutations in the DNA binding domain of p53 protein. Some of these changes lead not only to loss of wtp53 function but also mtp53 proteins that acquire novel Gain-of-Function (GOF) abilities. Our group showed that mtp53 associates with MCM2-7 and PARP1 on chromatin and that GOF mtp53 R273H associates with replicating DNA. What is not clear is which domain in R273H mtp53 is responsible for its chromatin-based replication activities. We hypothesize that GOF mtp53 proteins form tetramers in-vivo and this tetramer formation is critical for mtp53 chromatin-based activities. We used the glutaraldehyde crosslink assay to test mtp53 oligomerization in TNBC cell lines and saw that R273H, R248Q and R280K mtp53 exist predominantly as tetramers. We used the CRISPR/Cas9 system to delete amino acid residues 347-393 in a TNBC MDA-MB-468 cell line expressing R273H mtp53 (R273HÄ347-393). Using the glutaraldehyde crosslinking assay and western blot analyses, we demonstrated that R273HÄ347-393 clone expresses a truncated version of mtp53 and is a monomer. The R273HÄ347-393 cells grow at a slower rate than the parental derivative. We used site-directed mutagenesis to introduce three OD mutations which destabilize tetramer formation of mtp53 in Li-Fraumeni syndrome patients, in plasmids expressing either wtp53 or R273H mtp53. We will test if the OD influences GOF mtp53 chromatin-based activities by determining if dual mtp53-OD variants no longer interact with replicating DNA. Our findings will increase understanding of the roles played by the OD in GOF mtp53 activities.
Email questions and comments about this abstract to gannor@genectr.hunter.cuny.edu.
