Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences

Abstract

Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53R273H in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53R273H targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin.

Mutant p53 is a transcriptional co-factor that binds to G-rich regulatory regions of active genes and generates transcriptional plasticity

“…Furthermore, only active genes were regulated, as these genes were transcribed before and after mutp53 depletion. Interestingly, PPARGC1A and FRMD5, two genes we have previously described as mutp53 target genes in U251-derived cell clones with stable mutp53 reduction 21 were only slightly regulated 96 h after transient mutp53 depletion (1.92-and 1.55-fold respectively, Fig. S6F), indicating varying kinetics of transcriptional regulation after mutp53 depletion.…”

“…The cell growth advantage conferred by mutp53 is even more pronounced upon stable depletion. On an example of a cell clone derived from the U251 cells stably transfected with p53-specific shRNA, 21 we observed significant decrease in soft agar cloning efficiency (Fig. S6D) and propagation of neurospheres (Fig.…”

“…The probes on the tiling array were designed to cover whole genes including 1,500 nt upstream and downstream of transcriptional start and stop sites. As we have previously observed a significant binding of mutp53 to repetitive DNA, 21 we anticipated a relatively low sequence specificity and, consequently, high variability of mutp53 binding to gene regions. Surprisingly, however, we observed significant reproducibility of mutp53 binding in four biological replicates, e.g., in the mutp53 target gene EGR1 22 (Fig.…”

“…On the basis of a small set of mutp53 binding sites, we previously proposed that mutp53 modulates transcription on a global scale via direct binding to intronic and intergenic sequences prone to form non-B DNA structures. 21 To explore binding of mutp53 to genomic DNA in more detail, we performed the specificity of the interaction of mutp53 with DNA folded into a quadruplex structure. In addition, significant binding of mutant p53 (R248W and R273H mutations) was detected by EMSA using another G-rich oligonucleotide (TERT-Pu61) derived from of mutp53 proteins (G245S, R248W and R273H mutations) as well as of wtp53 to the quadruplex structure of the Pu52 oligonucleotide ( Fig.…”

“…20 Along with the binding of mutp53 to intronic and intergenic sequences enriched in repetitive DNA and prone to form non-B DNA structures, these data led to a model that attributes mutp53 GOF to its ability to modulate transcription on a global level via binding to structured DNA. 21 Both scenarios are complementary rather than mutually exclusive; however, data that incorporate both aspects of transcriptional regulation by mutp53 into one model are still insufficient.…”