The Role Of The Transcription Factor p53 In Transcription Reprogramming During HRAS-Mediated Breast Carcinogenesis

Tumorigenesis is a multistep process that is driven in part by alterations in gene expression programs which are critical to establish tumorigenic capacities.

We modeled cancer transformation by introducing an oncogenic copy of HRAS carrying a single point mutation at amino acid 12 (G12V) into MCF10A cells. This induced cancerous morphological and phenotypic changes in the transformed cells.

My goal is to understand the mechanistic basis of the transcriptional reprogramming in the transformed cells.

H-RAS is a small G protein which cannot affect directly the transcription program, yet RNA-seq analysis uncovered that more than thousand genes (>1.3 fold, p<0.05)were up or down regulated in the transformed cells. Interestingly ingenuity analysis indicated that many of the differentially expressed genes are targets of the tumor suppressor p53. Unexpectedly however, p53 protein and RNA levels did not alter in the transformed cells. To examine if p53 indeed plays a different regulatory role in the parental and transformed cells we stabilized p53 by nutlin-3a. This led to increase in the transcript levels of many known p53 targets such as MDM2. In addition RNA-seq analysis of nutlin-3a treated cells confirmed that the transformation-altered genes are indeed p53 targets.

To understand why p53 may have different gene targets in both cell types we asked whether p53 induces different changes in chromatin accessibility measured by ATAC-seq. This could also serve as indirect measure of p53 binding to chromatin. Global analysis of the cumulative accessibility signal at accessible sites that contain p53 motif showed that the accessibility was increased after nutlin-3a treatment, and to a greater extent in the parental cells. In addition, the signal at accessible sites with p53 motifs that are near differentially expressed genes is stronger than the signal at regulatory sites that are not close to differentially expressed genes. Here again, the effect is greater in the parental cells. This may suggest that the binding of p53 is facilitated by different factors in both cell lines.

Although p53 protein and RNA level in the transformed cells did not change, both chromatin binding and gene targets show different level, suggests that p53 indeed involve in the transcription reprogramming in the transformed cells.