NELF complex fosters BRCA1 and RAD51 recruitment to DNA damage sites and modulates sensitivity to PARP inhibition
Introduction
DNA double-strand breaks (DSBs) jeopardize genomic integrity and if left unrepaired, may trigger apoptosis or cellular transformation. DSBs are repaired by either homology-directed repair (HDR), an error free process that occurs at late S and G2, or by non-homologous end joining (NHEJ), an error prone pathway that is active throughout the cell cycle [1,2]. Unexpectedly, a plethora of recent reports demonstrated that the eukaryotic genome consists of hundreds of programmed (also called physiological) double-strand breaks (DSBs), distributed across the genome, playing a central role in activating gene expression [[3], [4], [5], [6], [7]]. Beside those physiological DSBs, intrinsic and extrinsic DNA damaging agents could cause unprogrammed DSBs that trigger local transcriptional silencing, plausibly to prohibit illegitimate transcription from broken DNA. Local DSB-induced transcriptional silencing is accomplished by establishing a transcriptionally restrictive chromatin environment, and by direct inhibition of RNA Pol II [[8], [9], [10], [11], [12], [13]]. Previously, we have shown that the negative elongation factor (NELF) complex interacts with RNA Pol II at DSB sites to shutdown transcription activity [10]. Furthermore, we demonstrated that NELF complex is required for timely repair of DSBs through a yet unknown mechanism. NELF complex consists of four subunits, NELF-A, NELF-E, NELF-B and NELF-C/D [14] and only NELF-E and NELF-A subunits are recruited to DSB sites [10]. Recently, it was shown that NELF-E exhibits oncogenic activity, as it promotes pancreatic cancer metastasis [15] and hepatocellular carcinomas (HCC) by regulating the stability and the transcription of MYC-target genes [16].
Here, we show that NELF complex promotes ionizing radiation-induced foci (IRIF) of BRCA1 and RAD51 and its depletion impairs HDR of DSBs in HCC cell line, Hep3B. Consequently, NELF-E depletion sensitizes BRCA1-proficient Hep3B cells to PARP inhibition. Altogether, we propose that the poor recruitment of BRCA1 and RAD51 to DSB sites in NELF-E-deficient cells may explain the defective HDR phenotype. Also, our data identify NELF-E and PARP1 as a novel synthetic lethal pair.
Section snippets
Cell lines
Hep3B and MCF7 cell lines were cultured in Dulbecco's modified Eagle medium (Gibco) and RPMI-1640 medium (Gibco), respectively. All cell lines were supplemented with 10 % heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine, 100unit/mL penicillin and 100 μg/mL streptomycin.
Cell irradiation and drug treatment
Cells were exposed to ionizing radiation from an X-ray machine (CellRad). Where indicated, cells were treated with ATM inhibitor (KU-60019), PARP inhibitor (Ku-0059436) and Caffeine (C8961; Sigma).
Western blot
Protein extracts
Results and discussion
We and others have shown that depletion of one NELF subunit (e.g. NELF-E) leads to a severe degradation of the remaining NELF subunits, NELF-A, NELF-B and NELF-C/D [10,20]. Because NELF-B is known to interact with BRCA1 (also called COBRA1, cofactor of BRCA1) [21], we wanted to determine whether NELF-E is also associated with BRCA1. Co-immunoprecipitation experiment shows that NELF-E interacts with BRCA1, irrespective of DNA damage inflicted by ionizing radiation (Fig. 1A). Next, we sought to
Author contributions
LAB performed the experiments described in Figs. 1A, B, H and 2 and S1−3. FEM performed the experiments described in Fig. 1C–E and helped in proofreading the manuscript and revising it. N.A. conceived the study, planed the experiments and wrote the manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
We thank Daniel Durocher for the plasmid encoding EGFP-BRCA1, Atsushi Miyawaki for the plasmid encoding EGFP-Geminin, and Tomer Shlomi for the generous gift of Hep3B cell line. We are grateful to Graham Dellaire for providing the pCR2.1-CloverPML and p X 330-LMNA-gRNA1 plasmids for the homologous recombination assay. Research in the Ayoub lab is supported by grants from the Israel Science Foundation (2511/18 & 2511/19) and from the Israel Cancer association (20200080).
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These authors contribute equally to this work.