Molecular Delineation Of Histone Acetyl Transferase TIP60 Dynamics And Plasticity

Covalent modifications of SUMO attachment is a rapid, energeticallyinexpensive mechanism for reversibly altering protein functions. Through a series ofenzyme-dependent biochemical cascade reaction, SUMO covalently attach to specificlysine(s) on substrate for exerting diverse biological significance, includingtranscriptional regulation, signal transduction, DNA damage repair, genomic integrityand chromatin remodeling.A fundamental question in cell biology is how cell maintains its genomicstability in response to genotoxic stress such as UV irradiation. Although mountingevidence demonstrates that the histone acetyltransferase TIP60 regulates the DNAdamage response following genotoxic stress by acetylating histone and remodelingchromatin, the molecular mechanisms underlying the TIP60-dependent response toUV-induced DNA damage remain poorly understood. To systematically analyzeproteins that regulate TIP60 activity during UV irradiation, we performed a proteomicanalysis of proteins selectively bound to "active" TIP60 using mass spectrometry andidentifed a novel SUMOylation dependent regulatory network underlyingUV-induced DNA damage response. Our biochemical characterization demonstratedthat TIP60 is SUMOylatable by SUMO-1 attachment on K430 and K451 in itsC-terminus in response to UV irradiation. This SUMOylation targets TIP60 to thePML nuclear bodied. Interestingly, the SUMOylation recruits p53 to the nuclearbodies and enables the trans-activation of its target genes. As a result, p21 proteinexpression is elevated in a TIP60-SUMOylation-dependent manner, which induces aG1 arrest in response to UV irradiation. Inhibition of TIP60 SUMOylation abrogatesthe p53-dependent DNA damage response while SUMOylation of TIP60 augments itsacetyltransferase activity in vitro and in vivo. Significantly, thisSUMOylation-amplified HAT activity of TIP60 induces ATR activation cascade. Thismechanism provides an explanation for how TIP60 links the UV-irradiated DNAdamage response signaling to DNA lesion repair.Though it is well established that TIP60 is a ubiquitous substrate and its degradation is inhibited following UV irradiation, the mechanism by which UVexposure modulates its stability is poorly understood. Here we show that the elevationof SUMOylation of TIP60 is consistent with the degradation inhibition of TIP60under UV irradiation. UV exposure facilitates the accumulation of SUMOylatedTIP60 in nucleoli and the accumulation is remarkedly promoted under UV andMG132 treatment, indicating SUMOylation functions to stabilize TIP60 in nucleoliby antagonizing its ubiquitination and nucleoli are potential locations in response toDNA lesion. UV irradiation upregulates TIP60 HAT activity, which subsequentlyactivates histone H2A acetylation and ATM PS1981, hence the phosphorylation ofp53 on Ser15 and Ser20 is evoked, indicating SUMO modification of TIP60 is anuniversal mechanism to activate ATM under various cellular stresses.The promyelocytic leukaemia (PML) protein is a major structural componentresponsible for PML nuclear bodies (PML NBs) integrity and modulating thefunctions of PML NBs in transcriptional regulation, tumor suppression and DNAdamage response. Although it is well defined that PML NBs are pools forSUMOylated proteins targeting, the mechanism by which PML protein regulates theprocess remains unclear. We recently discovered that the histone acetyltransfereaseTIP60 is novel SUMOylatable substrate and SUMO modification directs TIP60relocalization to PML NBs. Here we show that a specific PML isoform, PML3,stablizes TIP60 in PML NBs and the relocalization of TIP60 to PML NBs isdependent on the SUMOylation of PML3. PML3 physically interacts with TIP601～364aa region which is also responsible for TIP60 targeting to PML NBs. We alsodemonstrated that TIP60 specifies PML3 C-terminal 480～633aa association. Inaddition, we revealed that PML3 protects TIP60 from Mdm2-mediated ubiquitiousdegradation by competitively binding to TIP60 with Mdm2. Furthermore, wedemonstrated that TIP60 mediates PML3-modulated p53 stabilization in the presenceof Mdm2. These findings indicate a novel mechanistic association among PML3,TIP60, p53 and Mdm2 network for transcriptional regulation and cell grow control.