Gaëlle Legube

Tip60 is targeted to proteasome-mediated degradation by Mdm2 and accumulates after UV irradiation.

Acetylation is a prominent post‐translational modification of nucleosomal histone N‐terminal tails, which regulates chromatin accessibility. Accordingly, histone acetyltransferases (HATs) play major roles in processes such as transcription. Here, we show that the HAT Tip60, which is involved in DNA repair and apoptosis following γ irradiation, is subjected to proteasome‐dependent proteolysis. Furthermore, we provide evidence that Mdm2, the ubiquitin ligase of the p53 tumour suppressor, interacts physically with Tip60 and induces its ubiquitylation and proteasome‐dependent degradation. Moreover, a ubiquitin ligase‐defective mutant of Mdm2 had no effect on Tip60 stability. Our results indicate that Mdm2 targets both p53 and Tip60, suggesting that these two proteins could be co‐regulated with respect to protein stability. Consistent with this hypothesis, Tip60 levels increased significantly upon UV irradiation of Jurkat cells. Collectively, our results suggest that degradation of Tip60 could be part of the mechanism leading to cell transformation by Mdm2.

Tip60 and p400 are both required for UV-induced apoptosis but play antagonistic roles in cell cycle progression

The histone acetyl transferase Tip60 (HTATIP) belongs to a multimolecular complex involved in the cellular response to DNA damage. Tip60 participates in cell cycle arrest following DNA damage by allowing p53 to activate p21CIP (p21) expression. We show here that Tip60 and the E1A‐associated p400 protein (EP400), which belongs to the Tip60 complex, are also required for DNA damage‐induced apoptosis. Tip60 favours the expression of some proapoptotic p53 target genes most likely through the stimulation of p53 DNA binding activity. In contrast, p400 represses p21 expression in unstressed cells, thereby allowing cell cycle progression and DNA damage‐induced apoptosis. Tip60 and p400 have thus opposite effects on p21 expression in the absence of DNA damage. We further found that this antagonism relies on the inhibition of Tip60 function by p400, a property that is abolished following DNA damage. Therefore, taken together, our results indicate that Tip60 and p400 play distinct roles in DNA damage‐induced apoptosis and underline the importance of the Tip60 complex and its regulation in the proper control of cell fate.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

HIV‐1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP‐associated E4‐type ubiquitin‐ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60‐dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV‐1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.

Tip60 Acetyltransferase Activity Is Controlled by Phosphorylation

Here we show that the phosphorylation of histone acetyltransferase Tip60, a target of human immunodeficiency virus, type 1-encoded transactivator Tat, plays a crucial role in the control of its catalytic activity. Baculovirus-based expression and purification of Tip60 combined with mass spectrometry allowed the identification of serines 86 and 90 as two major sites of phosphorylation in vivo. The phosphorylation of Tip60 was found to modulate its histone acetyltransferase activity. One of the identified phosphorylated serines, Ser-90, was within a consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylatedin vitro by the cyclin B/Cdc2 complex. Accordingly, the phosphorylation of Tip60 was enhanced after drug-induced arrest of cells in G2/M. This G2/M-dependent phosphorylation of Tip60 was abolished by treating cells with a specific inhibitor of the cyclin-dependent kinase, roscovitin. All together, these results strongly suggest a G2/M-dependent control of Tip60 activity.

Deciphering the chromatin landscape induced around DNA double strand breaks

DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists. Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techniques that provide high-resolution maps of protein-genome interactions. However, applying these techniques to study chromatin changes induced around DSBs was previously hindered due to a lack of suitable DSB induction techniques. We have recently developed an experimental system utilizing a restriction enzyme fused to a modified oestrogen receptor ligand binding domain (AsiSI-ER), which generates multiple, sequence-specific and unambiguously positioned DSBs across the genome upon induction with 4-hydroxytamoxifen (4OHT) 1. Cell lines expressing this construct represent a powerful tool to study specific chromatin changes during DSB repair, enabling high-resolution profiling of DNA repair complexes and chromatin modifications induced around DSBs. Using this system, we have recently produced the first map of gH2AX, a DSB-induced chromatin modification, on two human chromosomes and have investigated its spreading properties 1. Here we provide additional data characterizing the cell lines, present a genome-wide profile of gH2AX obtained by ChIP-seq, and discuss the potential of our system towards investigations of previously uncharacterized aspects of DSB repair.

X-chromosome targeting and dosage compensation are mediated by distinct domains in MSL-3

In Drosophila, dosage compensation of X‐linked genes is achieved by transcriptional upregulation of the male X chromosome. Genetic and biochemical studies have demonstrated that male‐specific lethal (MSL) proteins together with roX RNAs regulate this process. Here, we show that MSL‐3 is essential for cell viability and that three domains in the protein have distinct roles in dosage compensation. The chromo‐barrel domain (CBD) is not necessary for MSL targeting to the male X chromosome but is important for male viability and equalization of X‐linked gene transcription. The polar region cooperates with the CBD in MSL‐3 function, whereas the MRG domain is responsible for targeting the protein to the X chromosome. Our results demonstrate that MSL‐3 localization to the male X chromosome and transcriptional upregulation of X‐linked genes are two separable functions of the MSL‐3 protein.

Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification

Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.