Elsevier

DNA Repair

Volume 97, January 2021, 103025
DNA Repair

NELF complex fosters BRCA1 and RAD51 recruitment to DNA damage sites and modulates sensitivity to PARP inhibition

https://doi.org/10.1016/j.dnarep.2020.103025Get rights and content

Highlights

Abstract

The negative elongation factor (NELF) is a four-subunit protein complex (NELF-E, NELF-A, NELF-B and NELF-C/D) that negatively regulates transcription elongation of RNA polymerase II (Pol II). Interestingly, upregulation of NELF-E subunit promotes hepatocellular carcinoma (HCC) and pancreatic cancer. In addition, we have previously shown that NELF complex fosters double-strand break (DSB)-induced transcription silencing and promotes homology-directed repair (HDR). However, the mechanisms underlying NELF-E regulation of HDR of DSBs remain unknown. Here, we show that NELF-E interacts with BRCA1 and promotes its recruitment to laser-microirradiated sites and facilitates ionizing radiation-induced foci (IRIF) of BRCA1 in HCC cells (Hep3B). The reduction in BRCA1 IRIF is accompanied by decreased RAD51 IRIF. A corollary to this, NELF-E-deficient Hep3B cells exhibit defective HDR of chromosomal DSBs induced by CRISPR-Cas9 system. Consequently, the disruption of NELF complex integrity, by NELF-E downregulation, sensitizes Hep3B cells to PARP inhibition. Altogether, our results suggest that NELF promotes HDR by facilitating BRCA1 and RAD51 IRIF formation and identify NELF complex as a novel synthetic lethal partner of PARP1.

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.

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