Tip60 Histone Acetyltransferase Activity Research Articles

Increasing Tip60 HAT Levels Rescues Axonal Transport Defects and Associated Behavioral Phenotypes in aDrosophilaAlzheimer's Disease Model

Axonal transport defects and axonopathy are prominent in early preclinical stages of Alzheimer's disease (AD), often preceding known disease-related pathology by over a year. As epigenetic transcriptional regulatory mechanisms, such as histone acetylation, are critical for neurogenesis, it is postulated that their misregulation might be linked to early pathophysiological mechanisms that contribute to AD. The histone acetyltransferase (HAT) Tip60 epigenetically regulates genes enriched for neuronal functions and is implicated in AD via its formation of a transcriptional regulatory complex with the amyloid precursor protein (APP) intracellular domain. Disruption of APP function is associated with axonal transport defects, raising the possibility that an epigenetic role for Tip60 might also be involved. Here, we examine whether Tip60 HAT activity functions in axonal transport using Drosophila CNS motor neurons as a well-characterized transport model. We show that reduction of Tip60 HAT activity in the nervous system causes axonopathy and transport defects associated with epigenetic misregulation of certain axonal transport-linked Tip60 target genes. Functional consequences of these defects are evidenced by reduced locomotion activity of the mutant Tip60 larvae, and these phenotypes can be partially rescued with certain histone deacetylase inhibitors. Finally, we demonstrate that Tip60 function in axonal transport is mediated by APP and that, remarkably, excess Tip60 exerts a neuroprotective role in APP-induced axonal transport and functional locomotion defects. Our observations highlight a novel functional interactive role between Tip60 HAT activity and APP in axonal transport and provide insight into the importance of specific HAT modulators for cognitive disorder treatment.

Epigenetic regulation of axonal growth of Drosophila pacemaker cells by histone acetyltransferase tip60 controls sleep.

Tip60 is a histone acetyltransferase (HAT) enzyme that epigenetically regulates genes enriched for neuronal functions through interaction with the amyloid precursor protein (APP) intracellular domain. However, whether Tip60-mediated epigenetic dysregulation affects specific neuronal processes in vivo and contributes to neurodegeneration remains unclear. Here, we show that Tip60 HAT activity mediates axonal growth of the Drosophila pacemaker cells, termed "small ventrolateral neurons" (sLNvs), and their production of the neuropeptide pigment-dispersing factor (PDF) that functions to stabilize Drosophila sleep-wake cycles. Using genetic approaches, we show that loss of Tip60 HAT activity in the presence of the Alzheimer's disease-associated APP affects PDF expression and causes retraction of the sLNv synaptic arbor required for presynaptic release of PDF. Functional consequence of these effects is evidenced by disruption of the sleep-wake cycle in these flies. Notably, overexpression of Tip60 in conjunction with APP rescues these sleep-wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for dTip60 in sLNv growth and function under APP-induced neurodegenerative conditions. Our findings reveal a novel mechanism for Tip60 mediated sleep-wake regulation via control of axonal growth and PDF levels within the sLNv-encompassing neural network and provide insight into epigenetic-based regulation of sleep disturbances observed in neurodegenerative diseases like Alzheimer's disease.

Tip60 HAT Activity Mediates APP Induced Lethality and Apoptotic Cell Death in the CNS of a Drosophila Alzheimer's Disease Model

Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. It has been demonstrated that Tip60 histone acetyltransferase (HAT) activity is involved in the transcriptional regulation of genes enriched for neuronal function as well as the control of synaptic plasticity. Accordingly, Tip60 has been implicated in the neurodegenerative disorder Alzheimer's disease (AD) via transcriptional regulatory complex formation with the AD linked amyloid precursor protein (APP) intracellular domain (AICD). As such, inappropriate complex formation may contribute to AD-linked neurodegeneration by misregulation of target genes involved in neurogenesis; however, a direct and causative epigenetic based role for Tip60 HAT activity in this process during neuronal development in vivo remains unclear. Here, we demonstrate that nervous system specific loss of Tip60 HAT activity enhances APP mediated lethality and neuronal apoptotic cell death in the central nervous system (CNS) of a transgenic AD fly model while remarkably, overexpression of Tip60 diminishes these defects. Notably, all of these effects are dependent upon the C-terminus of APP that is required for transcriptional regulatory complex formation with Tip60. Importantly, we show that the expression of certain AD linked Tip60 gene targets critical for regulating apoptotic pathways are modified in the presence of APP. Our results are the first to demonstrate a functional interaction between Tip60 and APP in mediating nervous system development and apoptotic neuronal cell death in the CNS of an AD fly model in vivo, and support a novel neuroprotective role for Tip60 HAT activity in AD neurodegenerative pathology.

Microarray Analysis Uncovers a Role for Tip60 in Nervous System Function and General Metabolism

BackgroundTip60 is a key histone acetyltransferase (HAT) enzyme that plays a central role in diverse biological processes critical for general cell function; however, the chromatin-mediated cell-type specific developmental pathways that are dependent exclusively upon the HAT activity of Tip60 remain to be explored.Methods and FindingsHere, we investigate the role of Tip60 HAT activity in transcriptional control during multicellular development in vivo by examining genome-wide changes in gene expression in a Drosophila model system specifically depleted for endogenous dTip60 HAT function.ConclusionsWe show that amino acid residue E431 in the catalytic HAT domain of dTip60 is critical for the acetylation of endogenous histone H4 in our fly model in vivo, and demonstrate that dTip60 HAT activity is essential for multicellular development. Moreover, our results uncover a novel role for Tip60 HAT activity in controlling neuronal specific gene expression profiles essential for nervous system function as well as a central regulatory role for Tip60 HAT function in general metabolism.

PDCD5 Interacts with Tip60 and Functions as a Cooperator in Acetyltransferase Activity and DNA Damage-Induced Apoptosis

Tip60 is a histone acetyltransferase (HAT) involved in the acetyltransferase activity and the cellular response to DNA damage. Here, we show that programmed cell death 5 (PDCD5), a human apoptosis-related protein, binds to Tip60 and enhances the stability of Tip60 protein in unstressed conditions. The binding amount of PDCD5 and Tip60 is significantly increased after UV irradiation. Further, PDCD5 enhances HAT activity of Tip60 and Tip60-dependent histone acetylation in both basal and UV-induced levels. We also find that PDCD5 increases Tip60-dependent K120 acetylation of p53 and participates in the p53-dependent expression of apoptosis-related genes, such as Bax. Moreover, we demonstrate the biological significance of the PDCD5-Tip60 interaction; that is, they function in cooperation to accelerate DNA damage-induced apoptosis and knockdown of PDCD5 or Tip60 impairs their apoptosis-accelerating activity, mutually. Consistent with this, PDCD5 levels increase significantly on DNA damage in U2OS cells, as does Tip60. Together, our findings indicate that PDCD5 may play a dual role in the Tip60 pathway. Specifically, under normal growth conditions, PDCD5 contributes to maintaining a basal pool of Tip60 and its HAT activity. After DNA damage, PDCD5 functions as a Tip60 coactivator to promote apoptosis.

PML3 Orchestrates the Nuclear Dynamics and Function of TIP60

The promyelocytic leukemia (PML) protein is a major component to govern the PML nuclear body (NB) assembly and function. Although it is well defined that PML NB is a site recruiting sumoylated proteins, the mechanism by which PML protein regulates the process remains unclear. Here we show that PML3, a specific PML isoform, interacts with and recruits TIP60 to PML NBs. Our biochemical characterization demonstrates that PML3 physically interacts with TIP60 via its N-terminal 364 amino acids. Importantly, this portion of TIP60 is sufficient to target to the PML NBs, suggesting that PML3-TIP60 interaction is sufficient for targeting TIP60 to the NBs. The PML3-TIP60 interaction is specific, since the region of TIP60 binding is not conserved in other PML isoforms. The physical interaction between PML3 and TIP60 protects TIP60 from Mdm2-mediated degradation, suggesting that PML3 competes with MDM2 for binding to TIP60. Fluorescence recovery after photobleaching analysis indicates that the PML3-TIP60 interaction modulates the nuclear body distribution and mobility of TIP60. Conversely, the distribution and mobility of TIP60 are perturbed in PML3-deficient cells, accompanied by aberrations in DNA damage-repairing response. Thus, PML3 orchestrates the distribution, dynamics, and function of TIP60. Our findings suggest a novel regulatory mechanism by which the PML3 and TIP60 tumor suppressors cooperate to ensure genomic stability.

Regulation of TIP60 by ATF2 Modulates ATM Activation

TIP60 (HTATIP) is a histone acetyltransferase (HAT) whose function is critical in regulating ataxia-telangiectasia mutated (ATM) activation, gene expression, and chromatin acetylation in DNA repair. Here we show that under non-stressed conditions, activating transcription factor-2 (ATF2) in cooperation with Cul3 ubiquitin ligase promotes degradation of TIP60, thereby attenuating its HAT activity. Inhibiting either ATF2 or Cul3 expression by small interfering RNA stabilizes the TIP60 protein. ATF2 association with TIP60 on chromatin is decreased following exposure to ionizing radiation (IR), resulting in enhanced TIP60 stability and activity. We also identified a panel of melanoma and prostate cancer cell lines whose ATF2 expression is inversely correlated with TIP60 levels and ATM activation after IR. Inhibition of ATF2 expression in these lines restored TIP60 protein levels and both basal and IR-induced levels of ATM activity. Our study provides novel insight into regulation of ATM activation by ATF2-dependent control of TIP60 stability and activity.

Stability of Nmi protein is controlled by its association with Tip60

Tip60 exerts diverse biological functions through mechanisms that are either dependent or independent on its intrinsic histone acetyltransferase activity. In the present study, we identified Nmi (N-Myc and STATs Interactor) as a novel binding partner for Tip60 by a yeast two-hybrid screen. The association of Tip60 with Nmi was further confirmed by coimmunoprecipitation in mammalian cells. The zinc finger domain of Tip60 interacts with the NID repeats of Nmi, a region essential for the cytoplamic localization and homo- and heterodimerization of Nmi. We further showed that Nmi is an unstable protein and is targeted for proteasome-mediated degradation. The stability of Nmi can be enhanced by its association with Tip60, a process that is dependent on histone acetyltransferase activity of Tip60. The stabilization of Nmi by Tip60 is in part mediated by the translocation of Tip60 into cytoplasm to form distinct large cytoplasmic speckles. Our finding that Tip60 stabilizes Nmi through the formation of distinct cytoplasmic speckles provides a new mechanism to modulate Nmi-mediated functions.

Serum Response Factor, an Enriched Cardiac Mesoderm Obligatory Factor, Is a Downstream Gene Target for Tbx Genes

We tested the idea that T-box factors direct serum response factor (SRF) gene activity early in development. Analysis of SRF-LacZ "knock-in" mice showed highly restricted expression in early embryonic cardiac and skeletal muscle mesoderm and neuroectoderm. Examination of the SRF gene for regulatory regions by linking the promoter and 5'-flanking sequences, up to 5.5 kb, failed to target LacZ transgene activity to the heart and the tail pre-somitic mesenchyme. However, linkage of a minimal SRF promoter with the SRF 3'-untranslated region (UTR), inundated with multimeric T-box binding sites (TBEs), restored robust reporter gene activity to embryonic heart and tail. Finer dissection of the 3'-UTR to a small cluster of TBEs also stimulated transgene activity in the cardiac forming region and the tail, however, when the TBEs contained within these DNA sequences were mutated, preventing Tbx binding, transgene activity was lost. Tbx2, Tbx5, and the cardiac-enriched MYST family histone acetyltransferase TIP60, were observed to be mutual interactive cofactors through the TIP60 zinc finger and the T-box of the Tbx factors. In SRF-null ES cells, TIP60, Tbx2, and Tbx5 were sufficient to stimulate co-transfected SRF reporter activity, however this activity required the presence of the SRF 3'-UTR. SRF gene transactivation was blocked by two distinct TIP60 mutants, in which either the histone acetyltransferase domain was inactivated or the Zn finger-protein binding domain was excised. Our study supports the idea that SRF embryonic cardiac gene expression is dependent upon the SRF 3'-UTR enhancer, Tbx2, Tbx5, and TIP60 histone acetyltransferase activity.

Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors and plays a critical role in regulating the expression of genes involved in androgen-dependent and -independent tumour formation. Regulation of the AR is achieved by alternate binding of either histone acetyltransferase (HAT)-containing co-activator proteins, or histone deacetylase 1 (HDAC1). Factors that control AR stability may also constitute an important regulatory mechanism, a notion that has been confirmed with the finding that the AR is a direct target for Mdm2-mediated ubiquitylation and proteolysis. Using chromatin immunoprecipitation (ChIP) and re-ChIP analyses, we show that Mdm2 associates with AR and HDAC1 at the active androgen-responsive PSA promoter in LNCaP prostate cancer cells. Furthermore, we demonstrate that Mdm2-mediated modification of AR and HDAC1 catalyses protein destabilization and attenuates AR sactivity, suggesting that ubiquitylation of the AR and HDAC1 may constitute an additional mechanism for regulating AR function. We also show that HDAC1 and Mdm2 function co-operatively to reduce AR-mediated transcription that is attenuated by the HAT activity of the AR co-activator Tip60, suggesting interplay between acetylation status and receptor ubiquitylation in AR regulation. In all, our data indicates a novel role for Mdm2 in regulating components of the AR transcriptosome.

Stabilization of Nmi Protein through Its Interaction with Tip60.

Tip60 (Tat-interactive protein of 60 kDa), a member of MYST family (for its founding members MOZ, YBF2/SAS3, SAS2 and Tip60) histone acetyltransferases, is involved in transcriptional regulation of many genes and diverse signaling transduction. The Tip60 complex consists of multiple subunits, with some implicated in DNA damage and apoptosis. The MYST superfamily members, such as MOZ and MORF, are implicated in the pathogenesis of acute myeloid leukemia. In the present study, Nmi was identified as a novel binding partner for Tip60 by a yeast two-hybrid screen. The association of Tip60 with Nmi was further confirmed by co-immunoprecipitation. Nmi, an interferon and interleukin inducible protein was originally identified as an N-Myc/c-Myc interacting protein. It is expressed in various adult and fetal tissues and in several cancer cell lines with the highest expression in certain myeloid leukemias. Nmi gene is localized on human chromosome 22q13.3 in a region where translocation has been reported in certain human leukemias. The Nmi protein interacts with all the STAT members except STAT2. Although Nmi lacks intrinsic transcriptional activation domain, it functions as an adaptor molecule to enhance STAT-dependent transcription through the recruitment of coactivator CBP/p300. Tip60 interacts with the C-terminus of Nmi in a region essential for Nmi-Nmi homodimerization and Nmi-IFP35 (interferon-inducible protein of 35 kDa) heterodimerization and subcellular localization. Homodimerization of Nmi reduces the interaction of Tip60 with Nmi. Nmi associates with the zinc finger domain of the Tip60, which is important for the transcriptional repression effect of Tip60 on TEL, the translocation E26 transforming-specific (ETS) leukemia. We further showed that Nmi is an unstable protein and is targeted for proteosome-mediated degradation possibly through ubiquitination of its C-terminus. The interaction with Tip60 enhances Nmi stability, which is dependent on the histone acetyltransferase activity of Tip60. Our results suggest that stabilization of Nmi protein through its interaction with Tip60 may be involved in the pathogenesis of certain leukemias.

Tip60 acetylates six lysines of a specific class in core histones in vitro.

Tip60, an HIV-1-Tat interactive protein, is a nuclear histone acetyltransferase (HAT) with unique histone substrate specificity. Since the acetylation of core histones at particular lysines mediates distinct effects on chromatin assembly and gene regulation, the identification of lysine site specificity of the HAT activity of Tip60 is an initial step in the analysis of its molecular function. Tip60 significantly acetylates amino-terminal tail peptides of histones H2A, H3 and H4, but not H2B, consistent with substrate preference on intact histones. Preferred acetylation sites for Tip60 are the Lys-5 of histone H2A, the Lys-14 of histone H3, and the Lys-5, -8, -12, -16 of histone H4, determined by a method which combined matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements and Lys-C endopeptidase digestion, or a method detecting the incorporation of radiolabelled acetate into synthetic peptides. The lysine site specificity of Tip60 in histone amino-terminal tail peptides in vitro has been characterized by an assay measuring the molecular mass of endopeptidase digested peptides, or a previously described assay. These results agree well with our proposed classification of lysines in core histones. The classification may be useful for an analysis of the relationships between HATs and the substrates of other uncharacterized HATs.