REGULATION OF GENOME INTEGRITY BY UBIQUITIN AND UBIQUITIN-LIKE MODIFICATIONS

The focus of our research is to investigate the role of ubiquitination and ubiquitin-like modifications in the regulation of different aspects of the DNA metabolism. We aim to understand how cells react to harmful conditions - genotoxic stress, inflammation, viral infection - by defining the ubiquitin and ubiquitin-like signature that marks chromatin in different cellular contexts, and by understanding how these ubiquitin-based signals are further decoded into functional outputs.

The stability of genetic information is undermined by a plethora of exogenous and endogenous agents inducing DNA damage. To preserve genomic integrity, cells evolved a process called “DNA Damage Response” or DDR, which activates cell cycle checkpoints and coordinates DNA repair, resulting in the hierarchical recruitment of DNA damage signaling and repair proteins to the site of lesion. If this repair process fails, the genome will introduce mutations or chromosomal translocations, leading to genomic instability, which is one of the most pervasive characteristics of human cancers. Moreover, as most commonly used therapeutic regimens are based on DNA damaging agents, DDR factors represent attractive targets to potentiate cancer chemotherapy, as recently exemplified by PARP inhibition.


THE UBIQUITIN SYSTEM

 Eukaryotic cells have developed efficient ways to modulate the properties of proteins, in order to rapidly respond to variations of external conditions and to face potentially dangerous external events. Among them is the reversible, covalent attachment of modifying groups. Post-translational modifications include small entities such as phosphate or acetyl group, but also entire protein, such as the member of ubiquitin (Ub) family. Ub is a 76 aminoacids polypeptide that has been found appended to the lysine residue of many proteins. A cascade of enzymes is required for the ubiquitination reaction, as depicted in Figure 1. After the first ubiquitin monomer, additional ubiquitin molecules can be attached to the target protein through any of the eight amine groups in the first molecule - the N-terminus (M1), K6, K11, K27, K29, K33, K48, and K63 - to form polyubiquitin chains. These different linkages increase the complexity of the ubiquitin system, giving rise to ubiquitin chains with distinct topology, providing structural flexibility that results in a multitude of functional outcomes. Although the roles of K48 and K63 polyUb in protein degradation and cellular signalling are well characterized, the functions of the remaining six non-canonical Ub chain types are still largely obscure.

(A) Ubiquitination is a versatile regulatory process that takes advantage of the combined action of 3 classes of enzymes: E1 activating enzyme, E2 conjugating enzyme and E3 ligase. (B) Ub is endowed with 7 different lysines, which can be substrate of further ubiquitination, giving rise to polyUb chains with different functional significance.


THE GENOME INTEGRITY IS HIGHLY REGULATED BY UBIQUITIN: ROLE OF RNF168

Ubiquitination is critical to preserve genome stability. Indeed, formation of DNA double strand breaks (DSBs) induced by genotoxic stress triggers a cascade of phosphorylation and ubiquitination events - the DDR - allowing the relaxation of chromatin structure and the recruitment of important downstream effectors, such as 53BP1 and BRCA1, which regulate DNA repair. Inhibition of these ubiquitin-mediated events has detrimental consequences for the cell.

A crucial role in this pathway is played by the Ub ligase RNF168, which ubiquitinates histones H2A and H2A.X and allows the activation of DDR (Pinato et al, BMC Mol Biol 2009; Pinato et al, MCB 2011). Inactivation of RNF168, either by point mutation or by depletion, completely blocks the pathway, increasing sensitivity of cells to genotoxic agents (Figure 2).

The relevance of ubiquitination, and in particular of RNF168, in the maintenance of genome stability has been directly demonstrated by the identification of two mutations in RNF168 gene as responsible of the RIDDLE (radiosensitivity, immunodeficiency, learning difficulties and dysmorphic features) syndrome, a recently described disorder characterised by cancer predisposition. These findings clearly indicate that chromatin ubiquitination, and RNF168 itself, play a pivotal role in the maintenance of genome stability and that its alteration may predispose to cancer development. Similarly, they suggest targeting of the ubiquitination pathway as chemo-potentiator of anticancer drugs.

(A) Schematic representation of RNF168: RF, RING finger domain; UMI, MIU1 and MIU2 are 3 Ub binding domains, UBDs. Ectopic expression of RNF168 induces extensive chromatin ubiquitination, as indicated by Ub immunoblotting (IB). Immunofluorescence analysis shows that RNF168 is recruited to the sites of DNA damage - called DDR foci – as revealed by co-localization with specific markers (phospho-H2A.X or γH2AX). (B and C) RNF168 is strictly required for proper activation of DDR: both point mutations addressing its Ub binding ability (UBDs*) and knock-down of the protein by shRNA targeting RNF168 result in the block of 53BP1 recruitment to DDR foci.


NON-CANONICAL UB MARK ON CHROMATIN SIGNALS DNA DAMAGE  

For long time it has been assumed that when cells are exposed to genotoxic agents, chromatin is modified by a specific type of modification, i.e. K63-linked ubiquitination, targeting H2A family of histones at a unique site at the C-terminal tail of histone H2A (on residue K119). Now, we added new mechanistic insights into Ub-based remodelling of chromatin, by demonstrating the following.

1) UbK13/K15: novel ubiquitination site on histone H2As induced by DNA damage. We discovered that lesions to DNA, instead of elongating Ub chain on K119, generate a novel Ub signature on the N-terminal tail of histone H2A and H2A.X (UbK13/K15), to transmit a specific signal to downstream effectors (Figure 3). This unprecedented Ub mark, generated by RNF168, is qualitatively different from the canonical UbK119 and warns the cell to activate DNA repair (Gatti et al, Cell Cycle 2012).

(A) Mass spectrometry analysis on chromatin extracts, derived from cells expressing RNF168, revealed a novel ubiquitination site on histone H2As, formed by K13/K15 residues. (B) This site is embedded in a highly conserved sequence. Mutation of the K13/K15 site maintains monoubiquitination of H2A (revealed by FLAG IB) but prevents further ubiquitination (Ub2, Ub3) induced by RNF168. On the contrary, a mutant affecting the canonical K118/K119 site is unable to form monoUb H2A in the absence of RNF168, as expected by theliterature, but it recovers full ubiquitination upon RNF168 expression.

2) RNF168 promotes K27-linked non-canonical ubiquitination to signal DNA damage.  Using a combined molecular biology techniques, biochemistry, specialized mass spectrometry analysis and a cell biology assay, we recently obtained unprecedented results showing that RNF168 activates the DDR by using K27-linked ubiquitination, and not K63 ubiquitination (Figure 4). We demonstrated that K27 ubiquitination is the major Ub-based modification that marks chromatin upon DNA damage and that it is strictly required for the proper activation of the DDR (Gatti et al, Cell Reports 2015).

a

(A) Schematic example of the Ub K-R mutants used in the experiment. Chromatin ubiquitination is highly impaired in cells expressing the K27R mutant of Ub, while displays only minor effects with other mutants. (B) Knock-down of Ub (siRNA Ub) completely blocks the recruitment of 53BP1 to the sites of lesion (see vector). DDR foci are restored by the expression of a siRNA-resistant form of Ub (WT-Ub), but not by the K27R mutant, clearly indicating a pivotal role of K27 linkage in this process (see quantification below).

These pioneering results open new perspectives in the field of genome stability, suggesting that specific epigenetic marks (i.e. alternative modification sites and novel Ub tags) can be used to signal detrimental cellular conditions. We just began to understand how chromatin remodels its structure by adding these bulky peptide-based tags, to generate a peculiar signal to alert the cell of a harmful situation. It is now essential drawing a clear picture of the dynamics of this novel modification that we discovered, in order to identify novel potential markers for diagnosis and therapy of human diseases.

WORKING MODEL

Formation of DSBs determined by genotoxic treatments induces a wave of phosphorylation events targeting different substrates – e.g. histone H2A.X - generating the docking sites for downstream DDR factors.

These events allow the recruitment of ubiquitinating enzymes, namely RNF8 and RNF168, which results in extensive ubiquitination of the surrounding chromatin.

Major targets of RNF168-induced ubiquitination are histones belonging to H2A family, which are modified on the new K13/K15 site at the N-terminal tail of the protein by a non-canonical type of ubiquitination, linked through K27 residue of Ub.

This Ub-mediated step is required for the recruitment to DDR foci of factors - 53BP1 and BRCA1 - responsible for activating downstream processes, including cell cycle arrest and DNA repair.