Abstract
Heterochromatin Protein 1 (HP1) and the Mre11-Rad50-Nbs1 (MRN) complex are conserved factors that play crucial role in genome stability and integrity. Despite their involvement in overlapping cellular functions, ranging from chromatin organization, telomere maintenance to DNA replication and repair, a tight functional relationship between HP1 and the MRN complex has never been elucidated. Here we show that the Drosophila HP1a protein binds to the MRN complex through its chromoshadow domain (CSD). In addition, loss of any of the MRN members reduces HP1a levels indicating that the MRN complex acts as regulator of HP1a stability. Moreover, overexpression of HP1a in nbs (but not in rad50 or mre11) mutant cells drastically reduces DNA damage associated with the loss of Nbs suggesting that HP1a and Nbs work in concert to maintain chromosome integrity in flies. We have also found that human HP1α and NBS1 interact with each other and that, similarly to Drosophila, siRNA-mediated inhibition of NBS1 reduces HP1α levels in human cultured cells. Surprisingly, fibroblasts from Nijmegen Breakage Syndrome (NBS) patients, carrying the 657del5 hypomorphic mutation in NBS1 and expressing the p26 and p70 NBS1 fragments, accumulate HP1α indicating that, differently from NBS1 knockout cells, the presence of truncated NBS1 extends HP1α turnover and/or promotes its stability. Remarkably, an siRNA-mediated reduction of HP1α in NBS fibroblasts decreases the hypersensitivity to irradiation, a characteristic of the NBS syndrome. Overall, our data provide an unanticipated evidence of a close interaction between HP1 and NBS1 that is essential for genome stability and point up HP1α as a potential target to counteract chromosome instability in NBS patient cells.
Highlights
Introduction The HeterochromatinProtein 1 (HP1) is a critical and conserved non-histone protein with essential roles in heterochromatin formation/maintenance and heterochromatin-related gene silencing[1]
By using extracts of HP1a-FLAG-expressing S2 cells, we found that HP1a is able to precipitate the endogenous MRN complex, indicating for the first time that HP1a binds all components of the complex (Fig. 1a)
While both GST-HP1aW200A and GSTHP1aI191E mutant proteins failed to efficiently pull down Nbs, the binding of endogenous Mre[11] and Rad[50] was influenced by the W200A mutation but only marginally by the I191E (Fig. 1c). This indicates that HP1a requires both CDS dimerization and a canonical binding interface to interact with Nbs, while its binding to Mre[11] and Rad[50] relies mainly on an intact C-terminal portion
Summary
Introduction The HeterochromatinProtein 1 (HP1) ( namedHP1a in Drosophila melanogaster and HP1α in Homo sapiens) is a critical and conserved non-histone protein with essential roles in heterochromatin formation/maintenance and heterochromatin-related gene silencing[1]. Official journal of the Cell Death Differentiation Association. Bosso et al Cell Death and Disease (2019)10:951. Mammalian HP1α accumulates at the site of DNA damage and loss of HP1α impairs the recruitment of RAD51, a key factor that promotes homologous recombination (HR) at double strand breaks (DSBs)[9]. Chromatin remodeling during HR repair is regulated by Meiotic Recombination 11 (MRE11), RAD50, and Nijmegen Breakage Syndrome 1 (NBS1; known as nibrin or NBN) (MRN) complex. This conserved complex allows the resection of damaged DNA and the docking of the complex with other DNA repair factors[11,12]
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