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Project 3. A role for FLASH/Casp8AP2 in R-loop regulation.

FLICE-associated huge protein (FLASH)/Caspase-8 associated protein 2 has been linked to various biological processes including death receptor-mediated signaling, transcriptional regulation via NF-kB and nuclear hormone receptors, Histone gene transcription, and Histone pre-mRNA processing of replication-dependent histone genes by interplay with CDK11. Our previous work identified FLASH as a nuclear body (NB)-associated protein localizing to PML NBs and Cajal bodies. Upon CD95 death receptor activation, FLASH shuttles from the cell nucleus to mitochondria where it potentiates caspase-8 activation, suggesting a nuclear amplification loop in CD95-mediated signaling. Furthermore, we contributed to the identification of the role of FLASH in histone gene transcription and found that FLASH protein levels are regulated by covalent SUMO modification regulating its degradation. Our unpublished work revealed that depletion of FLASH in mammalian cells using RNAi results in robust induction of g-H2AX foci, suggesting induction of DNA breaks. We hypothesized that FLASH depletion, by causing defects in transcriptional regulation and histone pre-mRNA processing, may result in increased R-loop formation and, in consequence, in DNA breaks. In line with our hypothesis, immunofluorescence staining using the RNA-DNA hybrid-specific S9.6 antibody showed a strong increase in nuclear, S9.6-positive signals in FLASH-depleted cells. Our project aims to elucidate the mechanism by which FLASH depletion triggers R-loop formation and to decipher the cellular response(s) triggered by increased R-loop formation.

Figure 1. Hypothetical model on the role of FLASH in R-loop regulation.

Along our hypothetical model on the role of FLASH in R-loop regulation (Figure 1), our project will address the following questions:

  1. Does R-loop formation upon FLASH depletion activate DNA damage checkpoint signaling?
  2. Is the R-loop-triggered DNA damage response functionally important for S-phase accumulation upon FLASH depletion?
  3. How does FLASH depletion trigger R-loop formation?
  4. Where in the genome are R-loops formed upon FLASH depletion?
  5. What is the biological consequence of increased R-loops in FLASH-depleted cells?
  6. Which proteins interact with FLASH, and do they phenocopy its effect on R-loops?