Recruitment of the cohesin loading factor NIPBL to DNA double-strand breaks depends on MDC1, RNF168 and HP1γ in human cells

doi:10.1016/j.bbrc.2011.07.021

Biochemical and Biophysical Research Communications

Volume 411, Issue 4, 12 August 2011, Pages 762-767

https://doi.org/10.1016/j.bbrc.2011.07.021 Get rights and content

Abstract

The cohesin loading factor NIPBL is required for cohesin to associate with chromosomes and plays a role in DNA double-strand break (DSB) repair. Although the NIPBL homolog Scc2 is recruited to an enzymatically generated DSB and promotes cohesin-dependent DSB repair in yeast, the mechanism of the recruitment remains poorly understood. Here we show that the human NIPBL is recruited to the sites of DNA damage generated by micro-irradiation as well as to the sites of DSBs induced by homing endonuclease, I-PpoI. The recruitment of NIPBL was impaired by RNAi-mediated knockdown of MDC1 or RNF168, both of which also accumulate at DSBs. We also show that the recruitment of NIPBL to the sites of DNA damage is mediated by its C-terminal region containing HEAT repeats and Heterochromatin protein 1 (HP1) interacting motif. Furthermore, NIPBL accumulation at damaged sites was also compromised by HP1γ depletion. Taken together, our study reveals that human NIPBL is a novel protein recruited to DSB sites, and the recruitment is controlled by MDC1, RNF168 and HP1γ.

Highlights

► NIPBL is recruited to DSBs. ► Localization of NIPBL to DSBs is regulated by MDC1 and RNF168. ► HP1γ is required for NIPBL accumulation at DSBs.

Introduction

Genome integrity is constantly challenged by DNA damage, resulting from a variety of genotoxic insults. DNA double-strand breaks (DSBs) are the most hazardous lesions because they can lead to cell death or cancer development if unrepaired or repaired incorrectly. DSBs trigger a conserved cellular response including DNA repair and cell cycle checkpoint to protect genome integrity. In fact, the defects of these cellular responses are observed in many tumor cells [1]. In response to DSBs, sensor proteins directly recognize damaged regions and recruit DNA damage response proteins to DSBs. The accumulation of these proteins occurs in a hierarchical fashion and is regulated by sequential signal transduction via protein–protein interaction and post-translational modifications. In human cells, DSBs are recognized by MRE11/RAD50/NBS1 complex, which triggers ATM-dependent phosphorylation cascade. At the vicinity of DSBs, ATM phosphorylates the variant histone H2AX. MDC1 binds directly to phosphorylated H2AX (γH2AX) via the BRCT domains, and then ATM-dependent phosphorylation of MDC1 recruits an E3 ubiquitin ligase, RNF8. RNF8-dependent histone ubiquitylation surrounding DNA lesions is further facilitated by another E3 ligase, RNF168. It is believed that such a DNA damage signaling cascade promotes further accumulation of proteins related to DNA repair or cell cycle checkpoint [2], [3].

Cohesin, which is composed of four evolutionarily conserved subunit SMC1, SMC3, Rad21 and SA1/2 in human cells, is a chromosome-associated protein and mediates cohesion between replicated sister chromatids [4]. In Saccharomyces cerevisiae, the loading of cohesin to chromosome depends on the heterodimeric cohesin loading factor, Scc2 and Scc4 [5]. Scc2 is evolutionally conserved from yeast to human [6], and the human Scc2 homolog NIPBL was identified as a responsible gene for about 60% individuals with Cornelia de Lange syndrome [7], [8]. Introducing mutation or depletion of homologs of NIPBL from yeast to human results in defects of sister chromatid cohesion [6], however the exact mechanism of cohesin loading to chromosomes by NIPBL is largely unknown.

Scc2/NIPBL has also a critical role in DNA damage response. In yeast, Scc2 is recruited to a HO endonuclease-induced DSB and facilitates DSB repair via Scc2-dependent accumulation of cohesin around the DSB [9], [10], [11]. In addition to Scc2, the loading of cohesin to the DSB requires DNA damage signaling factors, Tel1/Mec1 (orthologous to the mammalian ATM/ATR), phosphorylation of H2A (H2AX in mammals) and Mre11 (a subunit of MRE11/RAD50/NBS1 in mammals) [9]. In human, NIPBL was identified in comprehensive RNAi screenings for genes that are involved in sensitivity to ionizing radiation, and cells derived from Cornelia de Lange syndrome patients exhibited high frequency of chromosomal abnormalities following ionizing radiation [12], [13], [14]. However, unlike Scc2 in yeast, whether NIPBL accumulates around DSBs in human is unclear.

To address this question, we investigate NIPBL accumulation in response to DSBs in human cells. In this study, we show the recruitment of NIPBL to sites of DNA damage generated by UV-C micro-irradiation in the presence of BrdU or homing endonuclease I-PpoI. Furthermore, we also demonstrate that the recruitment of NIPBL depends on MDC1, RNF168 and HP1.

Section snippets

Cell culture

HT1080 cells and normal human diploid cells were cultured in a minimal essential Eagle’s medium supplemented with 10% fatal bovine serum (ThermoTrace Ltd.) at 37 °C in a humidified atmosphere containing 5% CO₂. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fatal bovine serum. HT1080 cells expressing MDC1 shRNA in a Doxycycline (Dox)-responsive manner were selected and cultured in a medium with 5 μg/ml puromycin. For generation of stable cell lines, HT1080

Recruitment of NIPBL to sites of DNA damage

To examine NIPBL accumulation at DNA damage sites in human cells, we applied UV-C irradiation through a micro-porous filter in the presence of photo-activating reagent BrdU to create local DNA damage [16], because we observed no recruitment of Halo-tagged NIPBL to DSBs generated by conventional γ-rays under the fluorescence microscopic technique (data not shown). Human fibrosarcoma cell line, HT1080, was transfected with the plasmid encoding Halo-tagged full-length NIPBL gene (Supplementary

Discussion

First, we investigated whether the cohesin loading factor NIPBL accumulation at sites of DSBs using conventional γ-rays, but no recruitment of NIPBL to the sites of DSBs was observed (data not shown). It may be due to the small number of accumulated NIPBL molecules in the close vicinity of DSBs. Another possibility is that the accumulation of NIPBL around DSBs may be buried under other NIPBL molecules which constitutively associate with chromatin even in unperturbed condition, because the

Acknowledgments

We are grateful to M.B. Kastan for providing the HA-ER-I-PpoI construct and T. Ogi for providing valuable reagents and critical reading of the manuscript. This work was supported by Global Center Of Excellence (GCOE) Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Recruitment of the cohesin loading factor NIPBL to DNA double-strand breaks depends on MDC1, RNF168 and HP1γ in human cells

Abstract

Highlights

Introduction

Section snippets

Cell culture

Recruitment of NIPBL to sites of DNA damage

Discussion

Acknowledgments

Mol. Cell

DNA Repair

Mol. Cell

Curr. Opin. Genet. Dev.

Mol. Cell

Mol. Cell

Curr. Biol.

Biochem. Biophys. Res. Commun.

Mol. Cell

Curr. Biol.

J. Mol. Biol.

Cell

Cell-cycle checkpoints and cancer

Nature

The cohesin complex and its roles in chromosome biology

Genes Dev.

Cornelia de lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster nipped-B

Nat. Genet.

NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de lange syndrome

Nat. Genet.