Measuring cell cycle-dependent DNA damage responses and p53 regulation on a cell-by-cell basis from image analysis

ABSTRACT

DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such damage is associated with the emergence of different cancers, neurological disorders and aging. DNA damage responses (DDR) in cells are closely associated with the cell cycle. While most of our knowledge of DDR comes from bulk biochemistry, such methods require cells to be arrested at specific stages for cell cycle studies, potentially altering measured responses; nor is cell to cell variability in DDR or direct cell-level correlation of two response metrics measured in such methods. To overcome these limitations we developed a microscopy-based assay for determining cell cycle stages over large cell numbers. This method can be used to study cell-cycle-dependent DDR in cultured cells without the need for cell synchronization. Upon DNA damage γH2A.X induction was correlated to nuclear enrichment of p53 on a cell-by-cell basis and in a cell cycle dependent manner. Imaging-based cell cycle staging was combined with single molecule P53 mRNA detection and immunofluorescence for p53 protein in the very same cells to reveal an intriguing repression of P53 transcript numbers due to reduced transcription across different stages of the cell cycle during DNA damage. Our study hints at an unexplored mechanism for p53 regulation and underscores the importance of measuring single cell level responses to DNA damage.

KEYWORDS:

• DNA damage response (DDR)
• cell cycle
• p53 regulation
• cell-by-cell correlation
• single molecule Fluorescence in situ Hybridization (smFISH)

Acknowledgements

We acknowledge the intramural research funds at TIFR Hyderabad to AM, and the TIFR graduate student fellowship to SD. SERB is acknowledged for an Early Career Research Award (ECR/2016/000907) to AM.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary data

Supplemental data for this article can be accessed here.

Additional information

Funding

This research was supported by intramural funds from Tata Institute of Fundamental Research (TIFR), and also partially by a Science and Engineering Research Board (SERB) Early Career Research Award (ECR/2016/000907) to AM.