An Epigenetic role for Tip60 in synaptic plasticity and neurodegenerative diseases

Age-associated cognitive decline and neurodegenerative disorders such as Alzheimers disease (AD) are associated with misregulation of synaptic plasticity linked genes; however the mechanisms underlying decline of such gene control during aging are unknown. Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. Accordingly, aberrant changes to histone acetylation patterns in the aging brain epigenome are linked to memory loss. It is therefore critical to identify and study the histone acetyltransferases (HAT) that create such marks. One such promising candidate is Tip60, a HAT important for various cellular processes and also implicated in AD and shown by our laboratory to be critical in regulating neuronal processes linked to cognition. To explore a direct role for Tip60 in synaptic plasticity, here we explore the consequences of misregulating Tip60 HAT activity in the Drosophila neuromuscular junction (NMJ). The Drosophila NMJ is an extremely well characterized, highly tractable and valuable tool to study synaptic plasticity. In addition, many of the pathways present at the Drosophila NMJ are well conserved and homologous to the mammalian CNS. We show that the HAT dTip60 is concentrated both pre- and post-synaptically within the NMJ. Presynaptic targeted reduction of dTip60 HAT activity significantly increases synaptic bouton number that specifically affects type Is boutons while postsynaptic reduction results in significant loss of these boutons. The excess boutons demonstrate defects in neurotransmission function. Analysis using immunohistochemical staining to the MAP, futsch reveals a significant increase in the rearrangement of microtubule loop architecture that is required for bouton division. Our results are the first to demonstrate a causative role for the HAT dTip60 in the control of synaptic plasticity that is achieved, at least in part, via regulation of the synaptic microtubule cytoskeleton. We also show its post- synaptic role in the muscles and its function in retrograde signaling in addition to anterograde mechanisms. We show that postsynaptic loss of Tip60 HAT activity affects DLG localization, leads to decrease in GluRIIC subunit localization thus suggesting roles in activity dependent mechanisms. We also demonstrate its role in regulating genes involved in activity dependent synaptic plasticity and wingless pathway. Our ChIP-qPCR data suggests regulation of these genes via acetylation of learning and memory marks, H3K9, H4K12 and H4K16. We also report the functional interaction between HAT deficient Tip60 and hAPP at the NMJ, pre- and post-synaptically via the intracellular domain of APP (AICD), the molecule implicated in AD. Presynaptic expression of APP/Tip60 double mutants cause drastic increases in bouton numbers, and decrease in active zone synaptic function marker bruchpilot suggesting defects in neurotransmission. Conversely, postsynaptic expression of the APP/Tip60 double mutants leads to marked decrease in bouton numbers and absence of GluRIIC and decrease in GluRIIB and GluRIIA receptor subunits suggesting defects in signaling mechanisms. These findings have implications for dTip60 HAT dependant epigenetic mechanisms in neurodevelopment and neurodenerative diseases.Ph.D., Biology -- Drexel University, 201

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

Background: Tip60 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 Findings: Here, 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. Conclusions: We 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

dTip60 HAT Activity Controls Synaptic Bouton Expansion at the Drosophila Neuromuscular Junction

Background: Histone acetylation of chromatin plays a key role in promoting the dynamic transcriptional responses in neurons that influence the neuroplasticity linked to cognitive ability, yet the specific histone acetyltransferases (HATs) that create such epigenetic marks remain to be elucidated. Methods and Findings: Here we use the Drosophila neuromuscular junction (NMJ) as a well-characterized synapse model to identify HATs that control synaptic remodeling and structure. We show that the HAT dTip60 is concentrated both pre and post-synaptically within the NMJ. Presynaptic targeted reduction of dTip60 HAT activity causes a significant increase in synaptic bouton number that specifically affects type Is boutons. The excess boutons show a suppression of the active zone synaptic function marker bruchpilot, suggesting defects in neurotransmission function. Analysis of microtubule organization within these excess boutons using immunohistochemical staining to the microtubule associated protein futsch reveals a significant increase in the rearrangement of microtubule loop architecture that is required for bouton division. Moreover, a-tubulin acetylation levels of microtubules specifically extending into the terminal synaptic boutons are reduced in response to dTip60 HAT reduction. Conclusions: Our results are the first to demonstrate a causative role for the HAT dTip60 in the control of synaptic plasticity that is achieved, at least in part, via regulation of the synaptic microtubule cytoskeleton. These findings have implication

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

Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE

<div><p>SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, <i>nse3</i>-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). <i>SIR4</i> recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, <i>nse3</i>-1/ <i>sir4</i> Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.</p></div

Viability analysis indicates genetic interaction between Tip60 and APP in <i>Drosophila</i>.

<p>The indicated transgene was expressed ubiquitously in the fly using 337-Gal4 driver or pan-neuronally using 179 y-Gal4 driver. The number of F1 progeny that eclosed were counted daily. The percentage of eclosed flies was calculated relative to the wild type control (<i>w¹¹¹⁸</i>). All crosses were carried out in triplicate at 25°C. Overexpression of APP drastically reduced viability to <10% while no effect was observed due to expression of truncated version of APP lacking its C-terminal domain. Overexpression of varying levels of wild type dTip60 (dTip60^WT) also reduced viability in a dose independent manner. However, co-expression of dTip60^WT with APP partially rescued the lethal effects induced by APP expression in a dose dependent manner with the maximum effect observed with high levels of dTip60^WT. In the presence of APP lacking the C-terminus, overexpression of dTip60^WT had similar effects seen in flies that overexpressed dTip60^WT alone.</p

Transgenic fly lines used for this study.

a<p>The Tip60 P-element insertion is located on chromosome 3 and the APP P-element insertion is located on chromosome 2.</p>b<p>Indicates where the transgenic fly lines were generated.</p

Smc5/6 is a telomere binding complex.

<p>(A) A schematic representation of the Smc5/6 complex showing the location of Nse3 as part of a trimeric sub-complex located at the head region where Smc5 and Smc6 meet. (B) Chromatin immunoprecipitation (ChIP) followed by qPCR was performed on Smc6^FLAG (JC1594) at the indicated time points after release from α-factor. The fold enrichment at three native subtelomeres (Tel1L, Tel6R and Tel15L) compared to a control (ctrl) late replicating region on Chromosome V (469104–469177) is reported with the mean ± SD for n≥3 experiments performed in technical duplicate. (*) Indicates a statistically significant level of enrichment compared to the ctrl with p values < .05 by a two-tailed <i>t</i>-test. Smc6^FLAG enrichment at Tel1L is higher at 0 and 15 minutes after release, but with p values = 0.08 and p = 0.06 respectively. The lower panels show flow cytometry on ChIP samples with an asynchronous culture shown in black at the 0 time point. (C) Drop assay of exponentially growing wild type (JC470) and <i>nse3</i>-1 (JC3607) cells that were grown for 48 hours at the indicated temperatures on YPAD and 1:5 serial dilutions. (D) Schematic diagram of Nse3. “MHD” represents <u>M</u>elanoma <u>H</u>omology <u>D</u>omain in Nse3 protein. Seven amino acid substitutions in Nse3-1 are shown in red. (E) Chromatin immunoprecipitation (ChIP) on Smc6^FLAG in wild type (JC1594), <i>nse3</i>-1 (JC2630), <i>mms21</i>-11 (JC2075) and the non-tagged (nt) control strains for wild type (JC470), <i>nse3</i>-1 (JC3607), and <i>mms21</i>-11 (JC1879) in asynchronous cultures. The fold enrichment levels are relative to the late-replicating control region on Chr V for n = 3 experiments with the mean ± SD. All primers are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.s002" target="_blank">S2 Table</a>. Enrichment levels for wild type and mutant cells with p values < .05 from a two-tailed <i>t</i>-test are indicated by (*). (F) Telomere length was determined as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref015" target="_blank">15</a>]. Southern blot analysis was performed on 1μg XhoI-digested genomic DNA hybridized with a radiolabeled poly (GT/CA) probe in wild type (JC471), <i>nse3</i>-1 (JC3032), <i>mms21</i>-11 (JC1981), and <i>smc6</i>-9 (JC1358).In higher eukaryotes, telomeres are challenged by the continuous loss of DNA due to the end replication problem. However, in <i>Saccharomyces cerevisiae</i>, telomere length is maintained by the continued expression of telomerase, an enzyme containing a RNA subunit that serves as a template for <i>de novo</i> telomere synthesis [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref016" target="_blank">16</a>]. After the 3’ end is extended by telomerase, the replicative DNA polymerase fills in the complementary strand. Both telomerase extension and semiconservative replication at telomeres are included in the final events of S phase (for review see [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref017" target="_blank">17</a>]). In the absence of telomerase activity, telomeres shorten extensively, leading to senescence, however a small percentage of cells survive by extending their telomeres through the HR dependent alternative lengthening of telomeres (ALT) pathway [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref018" target="_blank">18</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref021" target="_blank">21</a>].</p

Smc5/6 physically associate with Sir4 and is important for TPE.

<p>(A) Co-immunoprecipitation (Co-IP) as described in the materials and methods section was performed in cells carrying Sir4^Myc and Nse3^HA (JC3736) with Nse3^HA (JC2823) as control or (B) Sir4^Myc and Smc6^FLAG (JC3853) with Smc6^FLAG (JC1594) as a control. (C) ChIP was performed on Smc5^FLAG in wild type (JC3728) and <i>sir4</i>Δ (JC3720) and (D) Smc6^FLAG in wild type (JC1594) and <i>sir4</i>Δ (JC3732) and non-tagged (nt) strains in wild type (JC470) and <i>sir4</i> Δ (JC3737) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.g001" target="_blank">Fig 1E</a>. The fold enrichment levels are relative to the late-replicating control region on Chr V for n≥3 experiments with the mean ± SD at three native subtelomeres (Tel1L, Tel6R and Tel15L) with p values < .05 from a two-tailed <i>t</i>-test indicated. (E) TPE was determined in strains with the <i>URA3</i> reporter at the <i>adh4</i> locus of Chromosome VIIL. Tenfold (1:10) serial dilutions of overnight cultures were spotted onto SC (complete medium) and SC + .1% 5-FOA plates at 25°C and 34°C in wild type (JC1991), <i>sir4</i>Δ (JC3818), <i>nse3</i>-1 (JC3860), <i>mms21</i>-11 (JC1080) and <i>smc6</i>-9 (JC1077) isogenic strains.</p

Gene expression changes of dTip60^E431Q misregulated target genes in the different transgenic lines.

a<p>Quantitative RT-PCR analysis was performed for the indicated target genes.</p>b<p>Staged second instar larvae ubiquitously expressing the indicated transgene(s) were used for cDNA preparation. Quantitative RT-PCR reactions were carried out in triplicate and the relative fold change was calculated using the 2−ΔΔCT method using RP49 as control.</p>§<p>Genes that were differentially regulated between flies expressing the Tip60 HAT mutant dTip60^E431Q alone and in conjunction with APP.</p> <p>Gene that were differentially regulated between flies overexpressing dTip60^WT alone or together with APP.</p