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

Novel EAAT2 activators improve motor and cognitive impairment in a transgenic model of Huntington’s disease

IntroductionGlutamate excitotoxicity is causal in striatal neurodegeneration underlying motor dysfunction and cognitive deficits in Huntington’s disease (HD). Excitatory amino acid transporter 2 (EAAT2), the predominant glutamate transporter accounting for >90% of glutamate transport, plays a key role in preventing excitotoxicity by clearing excess glutamate from the intrasynaptic cleft. Accordingly, EAAT2 has emerged as a promising therapeutic target for prevention of neuronal excitotoxicity underlying HD and other neurodegenerative diseases.MethodsWe have previously designed novel EAAT2 positive allosteric modulator GT951, GTS467, and GTS551, with low nanomolar efficacy in glutamate uptake and favorable pharmacokinetic properties. In this study, we test the neuroprotective abilities of these novel EAAT2 activators in vivo using the robust Drosophila HD transgenic model expressing human huntingtin gene with expanded repeats (Htt128Q).ResultsAll three compounds significantly restored motor function impaired under HD pathology over a wide dose range. Additionally, treatment with all three compounds significantly improved HD-associated olfactory associative learning and short-term memory defects, while GT951 and GTS551 also improved middle-term memory in low-performing group. Similarly, treatment with GT951 and GTS551 partially protected against early mortality observed in our HD model. Further, treatment with all three EAAT2 activators induced epigenetic expression of EAAT2 Drosophila homolog and several cognition-associated genes.ConclusionTogether, these results highlight the efficacy of GT951, GTS467 and GTS551 in treating motor and cognitive impairments under HD pathology and support their development for treatment of HD

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

Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region

The five genes of the human growth hormone (hGH) cluster are expressed in either the pituitary or placenta. Activation of the cluster is dependent on a locus control region (LCR) comprising pituitary- specific (HSI,II, –15 kb), placenta-specific (HSIV, –30 kb) and shared (HSIII, –28 kb; HSV, –32 kb) DNase I hypersensitive sites. Gene activation in the pituitary is paralleled by acetylation of a 32 kb chromatin domain 5′ to the cluster centered at HSI,II. In the present study we observed that acetylation of this region in placental chromatin was discretely limited to shared HSIII and HSV. Transgenic studies revealed placenta-specific activation of linked genes by a determinant (P-element) located 2 kb 5′ to each of the four placentally expressed genes. A localized peak of histone acetylation was observed at these P-elements in placenta but not pituitary. These data support a model for bifunctional action of the hGH LCR in which separate positive determinants, HSI,II and the P-elements, activate their respective target genes by tissue-specific recruitment of distinctly regulated histone acetyl transferase activities

The Cloning and Characterization of the Histone Acetyltransferase Human Homolog Dmel\TIP60 in Drosophila melanogaster: Dmel\TIP60 Is Essential for Multicellular Development

Chromatin packaging directly influences gene programming as it permits only certain portions of the genome to be activated in any given developmental stage, cell, and tissue type. Histone acetyltransferases (HATs) are a key class of chromatin regulatory proteins that mediate such developmental chromatin control; however, their specific roles during multicellular development remain unclear. Here, we report the first isolation and developmental characterization of a Drosophila HAT gene (Dmel\TIP60) that is the homolog of the human HAT gene TIP60. We show that Dmel\TIP60 is differentially expressed during Drosophila development, with transcript levels significantly peaking during embryogenesis. We further demonstrate that reducing endogenous Dmel\TIP60 expression in Drosophila embryonic cells by RNAi results in cellular defects and lethality. Finally, using a GAL4-targeted RNAi system in Drosophila, we show that ubiquitous or mesoderm/muscle-specific reduction of Dmel\TIP60 expression results in lethality during fly development. Our results suggest a mechanism for HAT regulation involving developmental control of HAT expression profiles and show that Dmel\TIP60 is essential for multicellular development. Significantly, our inducible and targeted HAT knockdown system in Drosophila now provides a powerful tool for effectively studying the roles of TIP60 in specific tissues and cell types during development

Restoration of H4K5 and H4K12 acetylation levels in response to EE requires Tip60 HAT action in APP flies.

<p>The indicated transgene was expressed in the fly MB using GFP;;OK107-Gal4 driver. Expression of human APP in MB results no significant acetylation change in response to EE, while increased Tip60 HAT activity restores such response in acetylation of H4K5 and H4K12. (A) Representative immunoblot showing histone acetylation in WT, APP and APP;dTip60^WT flies under ISO and EE conditions. (B) Quantification of (A). Independent (unpaired) Student t-test was used to determine statistical significance between EE versus ISO conditions within the same genotype for each histone acetyl marks. Each blot was repeated at least three times. Each histone sample was extracted from fly heads. Proteins were extracted from at least two independent pooled tissue samples (30 heads for each extract). Each sample was run at least three times for statistic analysis. ** P < 0.01, *P < 0.05. Error bars indicate SEM.</p

Tip60 promotes EE neuroadaptative transcriptional benefits under APP induced neurodegenerative conditions.

<p>(A) Experiment schematic for the microarray analysis. (B) The hierarchical cluster of the 220 genes differentially regulated in response to EE in the wild-type control flies (corrected P-value < 0.05, FC> 1.3) reveals an impaired transcriptional response in Tip60 HAT mutant flies. (C) Pie diagram showing GO term associated enrichment in each of the major function categories identified in the analysis. (D) Number of genes that are up-regulated (dark gray) and down-regulated (light gray) in response to EE in control and Tip60 HAT mutant fly MB neurons with FC>1.1. (E) Function analysis on the selected gene group (highlighted on heatmap). This subset of genes is not significantly misregulated in comparison to WT under ISO condition, and is not responsive to EE induced neuroadaptive transcriptional benefits. (F) Quantitative RT-PCR validation of representative gene targets in control, APP and APP;dTip60^WT flies under ISO and EE conditions for EE-mediated transcriptional regulation on the representative genes. qPCR was performed using RNA isolated from fly heads. Histogram represents fold change in gene expression using ΔΔCt method with RP49 as the internal control. Student t-test was used to determine statistical significance between different housing conditions within the same genotype. ** P < 0.01, *P < 0.05. Error bars indicate SEM.</p