InteractiveFly: GeneBrief

Tip60: Biological Overview | References


Gene name - Tip60

Synonyms -

Cytological map position - 4A6-4B1

Function - enzyme

Keywords - acetyltransferase component of the Tip60 complex that promotes acetylation of histone H2A and the generation of silent chromatin - regulates sleep-wake cycle and axonal growth - mediator of APP induced lethality and apoptotic cell death - regulation of general metabolism and olfactory projection neuron dendrite targeting

Symbol - Tip60

FlyBase ID: FBgn0026080

Genetic map position - chrX:4,008,855-4,011,482

Classification - MYST-like histone acetyltransferase

Cellular location - nuclear and potentially cytoplasmic



NCBI links: Precomputed BLAST | Entrez Protein
Recent literature
Xu, S. and Elefant, F. (2015). Tip off the HAT- Epigenetic control of learning and memory by Drosophila Tip60. Fly (Austin) 9: 22-28. PubMed ID: 26327426
Summary:
Disruption of epigenetic gene control mechanisms involving histone acetylation in the brain causes cognitive impairment, a debilitating hallmark of most neurodegenerative disorders. Histone acetylation regulates cognitive gene expression via chromatin packaging control in neurons. Unfortunately, the histone acetyltransferases (HATs) that generate such neural epigenetic signatures and their mechanisms of action remain unclear. Recent findings provide insight into this question by demonstrating that Tip60 HAT action is critical for morphology and function of the mushroom body (MB), the learning and memory center in the Drosophila brain. This study shows that Tip60 is robustly produced in MB Kenyon cells and extending axonal lobes and that target MB Tip60 HAT loss results in axonal outgrowth disruption. Functional consequences of loss and gain of Tip60 HAT levels in the MB are evidenced by defects in memory. Tip60 ChIP-Seq analysis reveals enrichment for genes that function in cognitive processes and accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, increasing levels of Tip60 in the MB rescues learning and memory deficits resulting from Alzheimer's disease associated amyloid precursor protein (APP) induced neurodegeneration. These data highlight the potential of HAT activators as a therapeutic option for cognitive disorders.
Xu, S., Panikker, P., Iqbal, S. and Elefant, F. (2016). Tip60 HAT action mediates environmental enrichment induced cognitive restoration. PLoS One 11: e0159623. PubMed ID: 27454757
Summary:
Environmental enrichment (EE) conditions have beneficial effects for reinstating cognitive ability in neuropathological disorders like Alzheimer's disease (AD). While EE benefits involve epigenetic gene control mechanisms that comprise histone acetylation, the histone acetyltransferases (HATs) involved remain largely unknown. This study examine a role for Tip60 HAT action in mediating activity- dependent beneficial neuroadaptations to EE using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. Flies raised under EE conditions were shown to display enhanced MB axonal outgrowth, synaptic marker protein production, histone acetylation induction and transcriptional activation of cognition linked genes when compared to their genotypically identical siblings raised under isolated conditions. Further, these beneficial changes are impaired in both Tip60 HAT mutant flies and APP neurodegenerative flies. While EE conditions provide some beneficial neuroadaptive changes in the APP neurodegenerative fly MB, such positive changes are significantly enhanced by increasing MB Tip60 HAT levels. These results implicate Tip60 as a critical mediator of EE-induced benefits, and provide broad insights into synergistic behavioral and epigenetic based therapeutic approaches for treatment of cognitive disorder.
Panikker, P., Xu, S. J., Zhang, H., Sarthi, J., Beaver, M., Sheth, A., Akhter, S. and Elefant, F. (2018). Restoring Tip60 HAT/HDAC2 balance in the neurodegenerative brain relieves epigenetic transcriptional repression and reinstates cognition. J Neurosci. PubMed ID: 29654189
Summary:
Cognitive decline is a debilitating hallmark during pre-clinical stages of AD yet causes remain unclear. As histone acetylation homeostasis is critical for mediating epigenetic gene control throughout neuronal development, it was postulated that its misregulation contributes to cognitive impairment preceding AD pathology. This study shows that disruption of Tip60 HAT/HDAC2 homeostasis occurs early in the brain of an AD associated amyloid precursor protein (APP) Drosophila model and triggers epigenetic repression of neuroplasticity genes well before Abeta plaques form in male and female larvae. Repressed genes display enhanced HDAC2 binding and reduced Tip60 and histone acetylation enrichment. Increasing Tip60 in the AD associated APP brain restores Tip60 HAT/HDAC2 balance by decreasing HDAC2 levels, reverses neuroepigenetic alterations to activate synaptic plasticity genes, and reinstates brain morphology and cognition. Such Drosophila neuroplasticity gene epigenetic signatures are conserved in male and female mouse hippocampus and their expression and Tip60 function is compromised in hippocampus from AD patients. It is advocated that Tip60 HAT/HDAC2 mediated epigenetic gene disruption is a critical initial step in AD that is reversed by restoring Tip60 in the brain.
Giaimo, B. D., Ferrante, F., Vallejo, D. M., Hein, K., Gutierrez-Perez, I., Nist, A., Stiewe, T., Mittler, G., Herold, S., Zimmermann, T., Bartkuhn, M., Schwarz, P., Oswald, F., Dominguez, M. and Borggrefe, T. (2018). Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response. Nucleic Acids Res. PubMed ID: 29986055
Summary:
A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromatin and are implicated in the regulation of gene expression. This study shows that depletion of histone variant H2A.Z leads to upregulation of canonical Notch target genes and that the H2A.Z-chaperone TRRAP/p400/Tip60 complex physically associates with RBP-J at Notch-dependent enhancers. When targeted to RBP-J-bound enhancers, the acetyltransferase Tip60 acetylates H2A.Z and upregulates Notch target gene expression. Importantly, the Drosophila homologs of Tip60, p400 and H2A.Z modulate Notch signaling response and growth in vivo. Together, these data reveal that loading and acetylation of H2A.Z are required to assure tight control of canonical Notch activation.
Rust, K., Tiwari, M. D., Mishra, V. K., Grawe, F. and Wodarz, A. (2018). Myc and the Tip60 chromatin remodeling complex control neuroblast maintenance and polarity in Drosophila. Embo J. PubMed ID: 29997178
Summary:
Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. This question was addressed in Drosophila neural stem cells called neuroblasts. The Tip60 chromatin remodeling complex and its interaction partner Myc were identified as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. aPKC is the key target gene of Myc and the Tip60 complex subunit Domino plays a roll regulating neuroblast polarity. Transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. These findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division.

BIOLOGICAL OVERVIEW

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. This study shows 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, loss of Tip60 HAT activity in the presence of the Alzheimer's disease (AD) associated amyloid precursor protein (APP) was shown to affect 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 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 on sLNv growth and function under APP induced neurodegenerative conditions. These 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 (Pirooznia, 2012b).

Chromatin remodeling through histone-tail acetylation is critical for epigenetic regulation of transcription and has been recently identified as an essential mechanism for normal cognitive function. Altered levels of global histone acetylation have been observed in several in vivo models of neurodegenerative diseases and are thought to be involved in the pathogenesis of various memory related disorders. Chromatin acetylation status can become impaired during the lifetime of neurons through loss of function of specific histone acetyltransferases (HATs) with negative consequences on neuronal function. In this regard, the HAT Tip60 is a multifunctional enzyme involved in a variety of chromatin-mediated processes that include transcriptional regulation, apoptosis and cell-cycle control, with recently reported roles in nervous system function. Previous work has demonstrated that Tip60 HAT activity is required for nervous system development via the transcriptional control of genes enriched for neuronal function (Lorbeck, 2011). Tip60 HAT activity controls synaptic plasticity and growth (Sarthi, 2011) as well as apoptosis in the developing Drosophila central nervous system (CNS) (Pirooznia, 2012a). Consistent with these findings, studies have implicated Tip60 in pathogenesis associated with different neurodegenerative diseases. The interaction of Tip60 with ataxin 1 protein has been reported to contribute to cerebellar degeneration associated with Spinocerebellar ataxia (SCA1), a neurodegenerative disease caused by polyglutamine tract expansion (Gehrking, 2011). Tip60 is also implicated in Alzheimer's disease (AD) via its formation of a transcriptionally active complex with the AD associated amyloid precursor protein (APP) intracellular domain (AICD) (Cao, 2001; Slomnicki, 2008). This complex increases histone acetylation (Kim, 2004) and co-activates gene promoters linked to apoptosis and neurotoxicity associated with AD (Kinoshita, 2002). Additionally, misregulation of certain putative target genes of the Tip60/AICD complex has been linked to AD related pathology (Baek, 2002; Hernandez, 2009). These findings support the concept that inappropriate Tip60/AICD complex formation and/or recruitment early in development may contribute or lead to AD pathology via epigenetic misregulation of target genes that have critical neuronal functions. In support of this concept, it has been recently reported that Tip60 HAT activity exhibits neuroprotective functions in a Drosophila model for AD by repressing AD linked pro-apoptotic genes while loss of Tip60 HAT activity exacerbates AD linked neurodegeneration (Pirooznia, 2012a). However, whether misregulation of Tip60 HAT activity directly disrupts selective neuronal processes that are also affected by APP in vivo and the nature of such processes remains to be elucidated (Pirooznia, 2012b)

In Drosophila, the small and large ventrolateral neurons (henceforth referred to as sLNv and lLNv, respectively) are part of the well characterized fly circadian circuitry. Recent studied have implicated the l-and s-LNvs as part of the 'core' sleep circuitry in the fly, an effect that is predominantly coordinated via the neuropeptide pigment dispersing factor (PDF) that serves as the clock output, mediating coordination of downstream neurons. PDF is thought to be the fly equivalent of the mammalian neurotransmitter orexin/hypocretin because of its role in promoting wakefulness and thus stabilizing sleep-wake cycles in the fly. Within this circuit, the sLNvs are a key subset of clock neurons that exhibit a simple and stereotypical axonal pattern that allows high resolution studies of axonal phenotypes using specific expression of an axonally transported reporter gene controlled by the Pdf-Gal4 driver or by immunostaining for the Pdf neuropeptide that is distributed throughout the sLNv axons. These features make the sLNvs an excellent and highly characterized model neural circuit to study as they are amenable to cell type specific manipulation of gene activity to gain molecular insight into factors and mechanisms involved in CNS axonal regeneration as well as those that mediate behavioral outputs like sleep-wake cycle. Importantly, the Drosophila ventrolateral neurons (LNvs) have been previously used as a well characterized axonal growth model system to demonstrate that the AD linked amyloid precursor protein (APP) functions in mediating the axonal arborization outgrowth pattern of the sLNv (Leyssen, 2005). Based on these results, and previous studies reporting that Tip60 HAT activity itself is required for neural function (Lorbeck, 2011; Sarthi, 2011) and mediates APP induced lethality and CNS neurodegeneration in an AD fly model (Pirooznia, 2012a), It is hypothesized that APP and Tip60 are both required to mediate selective neuronal processes such as sLNv morphology and function that when misregulated, are linked to AD pathology. In the present study, this hypothesis was tested by utilizing the sLNvs as a model system to examine whether Tip60 mediated epigenetic dysregulation under neurodegenerative conditions such as that induced by APP overexpression leads to axonal outgrowth defects and if there is a corresponding effect on sLNv function in sleep regulation, a process that is also affected in neurodegenerative diseases like AD (Pirooznia, 2012b).

This report shows that Tip60 is endogenously expressed in both the sLNv and lLNvs. Specific loss of Tip60 or its HAT activity causes reduction of PDF expression selectively in the sLNvs and not the lLNv and shortening of the sLNv distal synaptic arbors which are essential for the pre-synaptic release of PDF from these cells. The functional consequence of these effects is evidenced by the disruption of the normal sleep-wake cycle in these flies, possibly through disruption of PDF mediated signaling to downstream neurons. By using transgenic fly lines that co-express full length APP or APP lacking the Tip60 interacting C-terminus with a dominant negative HAT defective version of Tip60, it was demonstrated that the APP C-terminus enhances the susceptibility of the sLNvs and exacerbates the deleterious effects that the loss of Tip60 HAT activity has on axon outgrowth and PDF expression. Importantly, these studies identify the neuropeptide PDF as a novel target of Tip60 and APP, that when misregulated results in sleep disturbances reminiscent to those observed in AD. Remarkably, overexpression of wild type Tip60 with APP rescues these sleep defects by increasing PDF expression and inducing overelaboration of the sLNv synaptic arbor area. Taken together, these findings support a neuroprotective role for Tip60 on sLNv growth and function under APP induced neurodegenerative conditions. The data also reveal a novel mechanism for PDF control via Tip60 and APP that provide insight into understanding aspects of sleep dependent mechanisms that contribute to early pathophysiology of AD (Pirooznia, 2012b).

Selective vulnerability of specific neuronal populations to degeneration even before disease symptoms are seen is a characteristic feature of many neurodegenerative diseases. Consistent with these studies, this study shows that when induction of the dTip60 RNAi response or expression of the dTip60 HAT mutant was directed to both the small and large LNvs, only the sLNvs were susceptible to the mutant effects induced under these conditions while the lLNvs were spared. The lack of any morphological effect on the lLNvs in the dTip60E431Q flies could stem from the fact that compared to the sLNvs, these neurons express higher levels of endogenous Tip60 that counteracts the mutant dTip60E431Q protein. However, induction of the RNAi response causes complete loss of Tip60 expression in both the lLNv and sLNv, and yet only the sLNvs are affected while the lLNv are spared, similar to the findings with dTip60E431Q expression. This suggests that the sLNvs may be more susceptible to misregulation of Tip60 or its HAT activity. Of note, the dTip60WT flies did not have any marked effect on the lLNv either, likely because these neurons are not susceptible to the moderate increase in Tip60 levels in the lLNvs induced under these conditions compared to the sLNvs. Developmentally, the sLNvs are known to differentiate much earlier than the large cells and this developmental difference may also in part account for the selective vulnerability of the sLNvs. In many neurodegenerative diseases, axon degeneration is known to involve protracted gradual 'dying-back' of distal synapses and axons that can precede neuron cell body loss and contribute to the disease symptoms. Importantly, loss of synapses and dying back of axons are also considered as early events in brain degeneration in AD. While APP overexpression in the LNvs did not have any observable effect on the sLNv axon growth at normal physiological temperatures, coexpression of the dTip60 HAT mutant with APP C-terminus appears to cause the sLNv axons in the adult animals to retract. The lack of any effect on the sLNv axon in the third instar larva in this case indicates that the axons grow to their full potential in the larval stage, but undergo degeneration post-mitotically in a process similar to 'dying-back' (Pirooznia, 2012b).

A functional interaction between Tip60 and the amyloid precursor protein (APP) intracellular domain (AICD) has been shown to epigenetically regulate genes essential for neurogenesis (Baek, 2002; Kinoshita, 2002; Pirooznia, 2012a). Such an effect is thought to be mediated by recruitment of the Tip60/AICD containing complex to certain gene promoters in the nervous system that are then epigenetically modified by Tip60 via site specific acetylation and accordingly activated or repressed. While the E431Q mutation in dominant negative HAT defective version of Tip60 (dTip60E431Q) reduces Tip60 HAT activity, it should not interfere with its ability to assemble into a protein complex (Yan, 2000; Lorbeck, 2011). Thus, dTip60E431Q likely exerts its dominant negative action over endogenous wild-type Tip60 via competition with the endogenous wild-type Tip60 protein for access to the Tip60/AICD complex and/or additional Tip60 complexes, with subsequent negative consequences on chromatin histone acetylation and gene regulation critical for nervous system function. This study shows that co-expression of HAT defective Tip60 (dTip60E431Q) with APP in the APP; dTip60E431Q flies exacerbates the mutant effects that either of these interacting partners has on the sLNv axon growth and Pdf expression when expressed alone. In contrast, co-expression of additional dTip60WT with APP alleviates these effects and this rescue is dependent upon the presence of the AICD region of APP. Thus, Tip60 HAT activity appears to display a neuroprotective effect on axonal outgrowth, Pdf expression, with concomitant alleviation of sleep defects under APP expressing neurodegenerative conditions. It is proposed that Tip60 might exert this neuroprotective function either by itself or by complexing with other peptides such as AICD for its recruitment and site specific acetylation of specific neuronal gene promoters to redirect their expression and function in selective neuronal processes such as sLNv morphology and function. Such a neuroprotective role for Tip60 is consistent with previous work demonstrating that excess dTip60WT production under APP expressing neurodegenerative conditions in the fly rescues APP induced lethality and CNS neurodegeneration and that dTip60 regulation of genes linked to AD is altered in the presence of excess APP (Pirooznia, 2012a). It is speculated that the degenerative effects observed in the APP; dTip60E431Q flies may result from formation of Tip60E431Q/AICD complexes that ultimately cause activation or de-repression of factors that promote axonal degeneration while excess Tip60/AICD complex formation in the APP;dTip60WT expressing flies promote gene regulation conducive for sLNv outgrowth and Pdf expression (Pirooznia, 2012b).

Sleep or wake promoting neurons in the hypothalamus or brainstem are known to undergo degeneration in a number of neurodegenerative diseases resulting in sleep dysregulation. In AD, such sleep disturbances are characterized by excessive daytime sleepiness and disruption of sleep during the night. These features resemble the symptoms of narcolepsy, a sleep disorder caused by general loss of the neurotransmitter hypocretin/orexin (Thannickal, 2009). Hypocretin is involved in consolidation of both nocturnal sleep and diurnal wake (Ohno, 2008) and loss of hypocretin levels have been correlated with sleep disturbances observed in AD (Fronczek, 2011). While the neuropathological changes in AD may contribute to hypocretin disturbances, a direct and causative role for APP in regulating hypocretin expression is not yet known. The LNv specific neuropeptide PDF is postulated to be the fly equivalent of hypocretin (Crocker, 2010) and has been shown to promote wakefulness in the fly. Consistent with these reports, the current data demonstrating somnolence during the light phase due to knock-down of PDF in the sLNv further supports a wake-promoting role for PDF. Accordingly, it was observed that overexpression of APP in the LNvs results in reduction of sLNv PDF expression as well as sleep disturbances that intriguingly, have been associated with AD pathology. The presence of similar effects on PDF and sleep due to loss of dTip60 HAT activity supports a role for both APP and Tip60 in controlling the PDF mediated sleep-wake regulation pathway. Previous studies have reported that the circadian modulators CLOCK and CYCLE regulate PDF expression in the sLNvs but not in the lLNvs. This study also observed a similar sLNv specific regulation of PDF by dTip60 in the adult flies. However, there was no effect on PDF expression in sLNvs in the larvae when Tip60 levels are undetectable. This is also consistent with the sLNv axonal defects that persist only in the adult flies. This suggests that the sLNvs may be subject to differential regulation during development as well as a temporal requirement for Tip60 in these cells in the adult flies. A recent study reported persistence of morning anticipation and morning startle response in LD in the absence of functional sLNv that were ablated due to expression of the pathogenic Huntington protein with poly glutamine repeats (Q128) (Sheeba, 2010). Consistent with the Sheeba study, this study did not observe any marked effect on the morning and evening anticipatory behavior in LD in the dTip60E431Q flies that exhibit a partial reduction in sLNv PDF. However, while the Q128 expressing flies were arrhythmic under constant darkness, dTip60E431Q flies maintain rhythmicity in DD indicating that the sLNvs are still functional in these flies. The remarkable cell specificity of PDF regulation indicates the presence of additional as yet unidentified clock relevant elements or developmental events that distinguish between the two cell types (Pirooznia, 2012b).

Recent evidence indicates that LNvs are light responsive and that their activation promotes arousal through release of PDF. Furthermore, PDF signaling to PDF receptor (PDFR) expressing neurons outside the clock, such as those found in the ellipsoid body that directly control activity, is thought to be important in translating such arousal signals into wakefulness (Parisky, 2008). Since PDF is released from the sLNv axon terminals, the retraction of the sLNv axon terminals induced by the Tip60 HAT mutant can interfere with PDF mediated interaction of the sLNvs with downstream circuits. In the case of APP overexpression, while sLNv axon structure is unaffected, PDF expression is reduced; it is speculated that the decrease in PDF under these conditions is responsible for the abnormal sleep phenotype observed. In support of this theory, it was found that expression of APP lacking the C-terminus that also has no observable effect on the sLNv axon growth or PDF expression did not have any effect on sleep behavior. Thus the results indicate that the degenerative effect on the sLNv axons and/or the effect on PDF expression could both contribute to the observed sleep disturbances. Likewise, co-expression of the dTip60 HAT mutant with full length APP or APP lacking the C-terminus affected both the sLNv axon growth and PDF expression and consequently resulted in similar sleep disturbances (Pirooznia, 2012b).

In addition to the wake promoting role, the LNvs also express GABAA receptors (Parisky, 2008; Chung, 2009) and are thus subject to inhibition by sleep promoting GABAergic inputs, analogous to those from the mammalian basal forebrain that regulate hypocretin neurons. The current consensus view is that sleep regulation is mediated by mutually inhibitory interactions between sleep and arousal promoting centers in the brain. The normal release of PDF from LNvs is part of the arousal circuitry in the fly and determines the duration of the morning and evening activity peaks while inhibition of these neurons and thus reduction in PDF release is necessary for normal sleep (Chung, 2009). Current models of sleep regulation suggest that the drive to sleep has two components, the first component is driven by the circadian clock and the second component is homeostatic in nature and the strength of this drive is based upon the amount of time previously awake. PDF release from sLNvs axon terminals exhibits diurnal variation and its release increases the probability of wakefulness by activating arousal promoting centers (Parisky, 2008). However, the homeostatic drive for sleep that accumulates during the wake period eventually inhibits such arousal centers to promote sleep. Consistent with these reports, the reduction of PDF observed due to either dTip60E431Q expression alone or co-expression of dTip60E431Q with APP that leads to flies sleeping more during the day may also lead to a decrease in their homoeostatic drive for sleep, thus resulting in the less consolidated sleep patterns observed for these flies during the night. Conversely, it was found that overexpression of sLNv PDF due to dTip60 overexpression induces wakefulness and arousal. Additionally, these flies exhibit impaired ability to maintain sleep at night that may be mediated through inappropriate activation of arousal circuits due to PDF overexpression. Similar effects have been reported in a Zebrafish model due to hypocretin overexpression that results in hyperarousal and dramatic reduction in ability to initiate and maintain a sleep-like state at night. Despite the moderate increase in sLNv PDF levels in the dTip60Rescue flies, no marked effect on sleep-wake cycle was observed in these flies. Extracellular levels of PDF and its signaling at synapses is thought to be regulated by neuropeptidases like neprilysin (see Neprilysin 4). In fact, neprilysin mediated cleavage of PDF has been shown to generate metabolites that have greatly reduced receptor mediated signaling (Isaac, 2007). Thus, it is speculated that the lack of any corresponding effect on sleep in the Tip60Rescue flies could be because such small increases in PDF might be regulated by endopeptidases like neprilysin. Based on these studies, a model is proposed by which the overelaborated sLNv synaptic arbors observed in flies co-expressing Tip60WT and APP may provide additional input sites for signals from sleep promoting neurons in the vicinity that counteract the arousing effect of PDF overexpression on nocturnal sleep (Pirooznia, 2012b).

Light mediated release of PDF from the lLNvs has been reported to modulate arousal and wakeful behavior as well as sleep stability. Thus, it has been suggested that the lLNvs may be part of an arousal circuit that is physiologically activated by light and borders with, but is distinct from the sLNvs and downstream sleep circuits (Sheeba, 2008). However, other studies have suggested that both LNv sub-groups promote wakeful behavior and that the lLNv act upstream of the sLNv (Parisky, 2008; Shang, 2008). The observation of sLNv directed effects on PDF expression and the persistence of sleep-wake disturbances suggest that the sLNvs may be part of the neural circuitry that regulates sleep downstream of the lLNvs via a PDF dependent mechanism. In this regard, the sLNvs may participate in the communication between the lLNvs and other brain regions to promote light mediated arousal. It has been proposed by that the lLNvs may promote neural activity of the Ellipsoid body (EB) in the central complex (CC), a higher center for locomotor behavior that expresses the PDF receptor. However, disruption of sleep-wake cycles was observed even in the absence of any marked effect on the lLNv morphology or PDF expression. While no direct projections from the lLNvs to the EB have been detected, the sLNv axonal projections are relatively closer to the CC and thus may promote PDF receptor mediated signaling in such regions that control activity. Sleep disturbances, while prominent in many neurodegenerative diseases are also thought to further exacerbate the effects of a fundamental process leading to neurodegeneration (Kang, 2009). For these reasons, optimization of sleep-wake pattern could help alleviate the disease symptoms and slow the disease progression. In this regard, the modulatory effects that Tip60 HAT activity (dTip60E431Q versus dTip60WT) has on the sLNvs, the fly counterpart of the mammalian pacemaker cells, under APP overexpressing conditions, may provide novel mechanistic insights into epigenetic regulation of neural circuits that underlie behavioral symptoms like the 'sundowners syndrome' in AD. Future investigation into the downstream mechanism by which Tip60 regulates the sleep-wake cycle may further provide insight into the utility of specific HAT activators as therapeutic strategies for sleep disturbances observed in AD (Pirooznia, 2012b).

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. This study demonstrates 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, this study shows that the expression of certain AD linked Tip60 gene targets critical for regulating apoptotic pathways are modified in the presence of APP. These 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 (Pirooznia, 2012a).

This study generated a unique transgenic Drosophila model system suitable for investigating a functional link between Tip60 HAT activity and APP in neuronal development, in vivo. Tip60 and APP were shown to functionally interact in both general and nervous system development in Drosophila, in vivo and that this interaction specifically mediates apoptotic neuronal cell death in the CNS, a process that when misregulated is linked to AD pathology. Remarkably, Tip60 appears to display a neuroprotective function in that Tip60 overexpression can rescue both loss of viability and neuronal apoptosis induction in a Drosophila AD model. While a number of in vitro studies supporting the transcription regulatory role of the Tip60/AICD complex in gene control have been reported, this work is the first to demonstrate a functional interaction between Tip60 HAT activity and APP in nervous system development in vivo (Pirooznia, 2012a).

This study shows that misexpression of Tip60 induces neuronal apoptotic cell death in the Drosophila CNS, and that this process is mediated via a functional interaction between Tip60 and the APP C-terminal domain. Since disruption of Tip60 HAT activity induced neuronal cell death, this study examined whether there was specific misregulation of apoptosis linked genes due to loss of Tip60 HAT activity. Pathway analysis of a previously reported microarray data set of genome wide changes in gene expression induced in the fly in response to Tip60 HAT loss (Lorbeck, 2011) revealed genes functioning in 17 different apoptotic pathways to be enriched, many of which were associated with the p53 apoptotic pathway. These findings are consistent with previous studies demonstrating a role for Tip60 as a p53 co-activator in p53 mediated apoptotic pathways. Recent studies have found Tip60 to be required for activation of proapoptotic genes through acetylation of p53 DNA binding domain. TRAF4, one such p53 regulated pro-apoptotic gene that responds to cellular stress was one of the genes that was found to be significantly upregulated in response to Tip60 HAT loss. The Myc family of transcription factors presents another instance of proteins involved in inducing apoptosis that are directly acetylated and stabilized by Tip60 and accordingly, Drosophila dMyc was found to be significantly upregulated in response to Tip60 HAT loss. Thus it is possible that the pro-apoptotic genes enriched in the dataset may represent both direct targets regulated by Tip60 epigenetic function as well as indirect targets of apoptosis regulators such as p53 that are controlled via their acetylation by Tip60. Misregulation of these pro-apoptotic genes in response to disruption of Tip60 HAT activity is also consistent with the observation that nervous system specific expression of dTip60E431Q induces apoptotic cell death in the CNS of dTip60E431Q larvae. This finding is in contrast to previous studies wherein cells expressing mutated Tip60 lacking HAT activity were reported to be resistant to apoptosis. However, these studies examined a role for Tip60 in DNA damage repair following cellular stress using the H4 neuroglioma cells in vitro. While Tip60 HAT activity is vital for DNA repair competency as well as for the ability to signal the presence of damaged DNA to the apoptotic machinery, how Tip60 HAT activity regulates differential gene expression profiles to prevent unwanted neuronal cell death during organismal development remains unclear. A number of mammalian studies have indicated that Tip60 can function not only as a coactivator, but also as a corepressor and as such, Tip60 has been shown to repress a vast array of developmental genes during ESC differentiation to maintain ESC identity. Consistent with these findings, the majority of pro-apoptotic genes identified that were misregulated in response to disruption of Tip60 HAT activity were upregulated, highlighting the crucial role Tip60 HAT activity plays in repression of apoptotic genes during neurogenesis that, when misregulated, likely contribute to dTip60E431Q induced apoptosis (Pirooznia, 2012a).

Interestingly, this study found that overexpression of wild type Tip60 in the nervous system also induced apoptosis in the CNS. Furthermore, overexpressing Tip60 was found to induce expression of pro-apoptotic genes such as ALiX and CalpA while downregulating others like Wingless, Frizzled and dMyc that have multiple essential functions during Drosophila development. These bidirectional gene expression changes suggest that increasing Tip60 mediated acetylation can also lead to complex changes in the chromatin landscape resulting in inappropriate activation and/or repression of apoptosis competent genes as well as those crucial for development. Accumulating evidence shows that hyperacetylation can be fatal to neurons. Under normal conditions, increasing hyperacetylation by treating neurons with a general HDAC inhibitor like trichostatin A has been found to induce neuronal apoptosis. Similarly, increasing acetylation levels by overexpressing the HAT CBP in resting neurons has been reported to enhance chromatin condensation and neuronal death. In order to maintain cellular homeostasis, HAT/HDAC equilibrium and therefore histone acetylation is strictly regulated as it is essential to maintain the functional status of neurons. Based on these findings, it is speculated that overexpression of Tip60 disrupts the acetylation balance, thus skewing the neuronal survival pathway towards apoptosis and ultimately cell death. In support of this concept, altered levels of global histone acetylation have been observed in many in vivo models of neurodegenerative diseases (Pirooznia, 2012a).

Another striking feature of the apoptotic microarray gene enrichment search was the identification of apoptosis linked pathways associated with neurodegenerative diseases like Parkinson's, Huntington's and Alzheimer's disease. These diseases are also characterized by neuronal cell death that increases over time and underlies an array of symptoms that depend on the function of the lost neuronal population. It has been proposed that in AD, in addition to the deposition of toxic β-amyloid plaques in the brain, neurodegeneration may also be caused via γ-secretase cleavage of APP that generates AICD carboxy terminal fragments that are toxic to neurons. Accordingly, ectopic expression of AICD in rat pheocytoma cells and cortical neurons has been shown to induce apoptosis upon nuclear translocation. Consistent with these reports, induction of apoptosis was observed when APP is expressed in the nervous system of Drosophila in vivo at physiological temperatures, and this phenotype is dependent upon the C-terminal domain of APP. Interestingly, APP C-terminal domain induced apoptosis has previously been reported to be mediated via Tip60 HAT activity in vitro, such that induction of apoptosis in neuroglioma cells transfected with APP C-terminal domain is enhanced by co-transfection of wild type Tip60 and decreased by a dominant negative version of Tip60 lacking HAT activity. In contrast, this study demonstrated that nervous system specific co-expression of APP and HAT defective mutant Tip60 increases apoptosis while overexpression of wild-type Tip60 with APP counteracts this effect and that these phenotypes are dependent upon the Tip60 interacting C-terminus of APP. Such differences may be accounted for by the fact that these studies were carried out in a developmental model system, in vivo. However, the effects this study has show on neuronal apoptosis are also consistent with the effects observed in the viability assay wherein lethality caused by neuronal overexpression of APP was enhanced by reduction of Tip60 HAT activity and suppressed by additional Tip60 levels. Importantly, this finding, in conjunction with previously published reports supporting a causative role for Tip60 in the control of synaptic plasticity (Sarthi, 2011) and the transcriptional regulation of genes enriched for neuronal function (Lorbeck, 2011), support the concept that misregulation of Tip60 HAT activity can lead to aberrant gene expression within the nervous system that contributes to the AD associated neurodegenerative process (Pirooznia, 2012a).

Tip60 has been implicated in AD via its transcriptional complex formation with AICD. Thus, experiments were carried to determine whether the expression of specific genes that are misregulated by dTip60E431Q or dTip60WT are modified by the presence of APP. Intriguingly, a number of these genes were found to be differentially regulated under APP expressing conditions. Two such genes, Wingless and Frizzled, which are upregulated in dTip60E431Q flies and repressed in dTip60WT flies are particularly interesting. Wingless, the Drosophila segment polarity gene and its membrane receptor Frizzled are known to be required for specification and formation of various neurons in the CNS and belong to the Wnt signaling pathway. In addition to Wingless and Frizzled being important for the disease process, they are also crucial for normal growth and development. Intriguingly, it was found that co-expressing APP with either the Tip60 HAT mutant or in the Tip60 overexpressing background has a repressive effect on these essential genes. Recent evidence supports a neuroprotective role for the Wnt signaling pathway and a sustained loss of Wnt signaling function is thought to be involved in aβ induced neurodegeneration. Drosophila Myc is a regulator of rRNA synthesis and is necessary for ribosome biogenesis during larval development and is another instance of a vital gene that exhibited reduced expression under APP expressing conditions. Thus misregulation of such developmentally required genes in conjunction with the other pro-apoptotic genes in the data set likely contributed to the observed enhanced apoptotic cell death in the CNS of APP;dTip60E431Q larvae. In contrast, this study found the Drosophila homolog of Bcl-2 protein, Buffy to be repressed in the APP; dTip60E431Q flies that displayed an increase in apoptosis. Consistent with the findings, recent studies have reported that Buffy has anti-apoptotic functions in vivo and intriguingly, this study found its expression to be significantly induced in the APP; dTip60WT flies that also exhibited a marked reduction in apoptosis induced cell death when compared to flies expressing dTip60WT alone. These findings suggest that induction of such pro-survival factors could mediate the dTip60 induced rescue of APP mediated defects that were observe in these flies (Pirooznia, 2012a).

Differential regulation of the microarray targets were found between flies that express dTip60E431Q alone and in conjunction with APP, in that the majority of genes tested are repressed in the APP;dTip60E431Q double mutants and activated in dTip60E431Q flies. These results indicate that the presence of APP can modulate the transcriptional regulatory potential of Tip60. The APP intracellular domain was recently shown to lower the sensitivity of neuronal cells to toxic stimuli and transcriptionally activate genes involved in signaling pathways that are not active under basal conditions). APP could mediate such effects either by sequestering Tip60 away from its typical target promoters or by displacing another factor in the complex that is also required for regulating transcription. Additionally, Tip60 has been shown to function as a negative regulator of gene expression. In fact, overexpression of Tip60 but not its HAT deficient mutant has been reported to function as co-repressor for gene repression mediated by transcription factors like STAT3 and FOX3, an effect that is mediated through association with specific histone deacetylases. This could partly account for the repressive effects that were observed due to overexpression of wild type Tip60 either alone or in conjunction with APP. Tip60 can also function as a co-activator of gene transcription via displacement of co-repressors on the promoters of specific genes. For instance, it has been reported that following IL-1 stimulation, recruitment of a wild type Tip60 containing co-activator complex leads to activation of p50 target genes like KAI1/CD82 through displacement of a specific NCoR co-repressor complex. Intriguingly, the Tip60-FE65-AICD containing complex was shown to similarly displace the NCoR complex and derepress such targets, suggesting a potential transcription activation strategy that underlies the gene expression changes observed under APP overexpressing conditions. Since loss of Tip60 HAT activity enhances APP induced lethal effects in the nervous system and overexpression of wild type Tip60 diminishes these defects, it is hypothesized that the Tip60-AICD containing complex may mediate these rescue effects either via regulation of a subset of gene targets different from those targeted by either APP or Tip60 alone or by differentially regulating the same gene pool such as that seen in the case of the anti-apoptotic gene Buffy. Thus, although the repertoire of genes that were tested include both mediators as well as inhibitors of apoptosis, taken together the data support a model by which Tip60 HAT activity plays a neuroprotective role in disease progression by complexing with the AICD region of APP to epigenetically regulate transcription of genes essential for tipping the cell fate control balance from apoptotic cell death towards cell survival under neurodegenerative conditions such as excess APP. Therefore, a neuroprotective role is proposed for Tip60 in AD linked induction of apoptotic cell death. Future investigation into the mechanism by which Tip60 regulates these processes may provide insight into the utility of specific HAT activators as therapeutic strategies for neurodegenerative disorders (Pirooznia, 2012a).

Microarray analysis uncovers a role for Tip60 in nervous system function and general metabolism

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. This study investigated 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. Amino acid residue E431 in the catalytic HAT domain of dTip60 is critical for the acetylation of endogenous histone H4 in the fly model in vivo, and dTip60 HAT activity is essential for multicellular development. Moreover, the 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 (Lorbeck, 2011).

To create a suitable in vivo model to exclusively explore the role of Tip60 HAT activity in developmental gene control during multicellular development, transgenic flies producing a dominant negative HAT defective version of Tip60 were created by introducing the amino acid substitution E431Q into its conserved catalytic HAT domain. Although the corresponding mutation in the Tip60 yeast homolog EsaI (E338Q) was shown to retain proper folding, and display a dominant negative effect on yeast cell growth by specifically disrupting EsaI HAT activity via putative disruption of the proton extraction capability of the enzyme, it was unknown whether the mutant dTip60 protein would display similar dominant negative effects in the multicellular model system of Drosophila. Production of dTip60E431Q in flies was shown to cause both a reduction in endogenous acetylated H4 histones in vivo and a dominant negative lethal effect with increasing severity correlating with higher levels of mutant dTip60E431Q. Based on these results, it is speculated that the mutant dTip60E431Q protein may produce its dominant negative effect in the fly by outcompeting endogenous wild-type dTip60 for recruitment to chromatin when over-expressed, thus titrating out endogenous histone H4 chromatin acetylation, with resultant deleterious effects on gene expression. Taken together, the findings support a critical role for dTip60 catalytic HAT residue E431 in the acetylation of histone H4 in vivo and show that dTip60 HAT activity is essential for multicellular development and are consistent with prior studies demonstrating and essential role for Tip60 in fly and mouse development. As Tip60 plays an important role in regulating apoptosis and double stranded break repair, lethality may result, at least in part, by defects in multiple cell division pathways. These findings support that the experimental system is a novel and valuable model for investigating the effects of epigenetic modifications, especially of the Tip60 HAT enzyme, on the developmental processes in vivo (Lorbeck, 2011).

Microarray analysis of the genome-wide gene changes that result in flies in response to HAT mutant dTip60E431Q production revealed that the majority of misregulated genes clustered into 17 significantly enriched groups, with 8 of these groups each linked to metabolic processes including amino acid, carbohydrate, lipid, glycoprotein and fatty acid metabolism. The significant enrichment of these Tip60 HAT affected metabolic genes supports a central role for Tip60 HAT function in general cellular metabolism. The findings are consistent with previous studies directly linking Tip60 in the epigenetic based transcriptional control of the central metabolic regulator LRP1 (Liu, 2007) a lipoprotein receptor essential for lipid and cholesterol metabolism. Tip60 also serves as a co-activator for the regulation of transcription factor peroxisome proliferator-activated receptor γ (PPARγ) target genes that play key roles in the regulation of lipid and glucose metabolism. Importantly, a recent elegant study using protein acetylation microarray analysis in yeast demonstrated that the NuA4 complex (yeast homolog of the human Tip60 complex), and specifically EsaI (yeast Tip60 homolog), controls the activity of the central glucose metabolism regulator phosphoenolpyruvate carboxykinase (Pck1p) via its direct acetylation (Lin, 2009). Based on this finding, it is speculated that the Tip60 HAT metabolic associated direct and indirect target genes that were identified in this study may not only be controlled epigenetically by Tip60 HAT action, but may also represent indirect targets of central metabolic regulator proteins that are directly controlled via their acetylation by Tip60. Of note, the majority of misregulated genes identified in response to dTip60 HAT depletion were upregulated, supporting a critical role for Tip60 HAT activity in the repression of target genes, possibly by the direct recruitment and interaction of Tip60 with transcriptional silencers and/or histone deacetylases that are dependent upon Tip60 acetylation for complex formation or via specific Tip60 chromatin acetylation marks that promote recruitment of such silencers to these genes, or by reorganization of chromatin into a repressive environment. Involvement of Tip60 in transcriptional repression is not unprecedented, with a previous study supporting a critical role for Tip60 in epigenetically repressing a large number of developmental genes essential for embryonic stem cell (ESC) differentiation (Fazzio, 2008). Additionally, expression of the yeast homolog of dTip60E431Q, HAT-defective dominant negative Esa1E338Q leads to transcriptional silencing of ribosomal DNA (rDNA) in yeast via reorganization of nucleolar chromatin structure (Clarke, 1999). Moreover, a recent microarray analysis of RNAi induced Tip60 knockdown in Drosophila embryonic S2 cell culture also revealed a significant portion of genes that were upregulated in response to loss of dTip60 activity (Schirling, 2010). Comparison of the current data with this previous study revealed 11 identical clusters between the two sets of upregulated gene data that included immune responses, transmembrane transport, cell adhesion, protein modification, morphogenesis and importantly, diverse metabolic processes and nervous system development. However, unlike the Schirling study, the current study did not identify strong enrichment of genes with chromatin-related annotations among the 'repressed' genes identified and only approximately 28% of the misregulated genes overlap. Such differences may be due to the different starting material (embryonic Drosophila cell culture versus whole larval preparation) and knockdown systems (RNAi versus dTip60E431Q) used in the Schirling and the current study, respectively. These differences are important as they suggest that dTip60 may regulate different sets of genes as development proceeds (Lorbeck, 2011).

Epigenetic regulation has been postulated to provide a coordinated system of regulating gene expression at each stage of neurogenesis, thus promoting brain and CNS development, neural plasticity, learning, and memory. The identification of a number of neurological disorders that result from HAT misregulation underscores a crucial role for acetylation in proper CNS development. For example, missense mutations in the CBP and p300 genes or loss of a CBP allele cause Rubinstein-Taybi syndrome (RTS), a human disease that displays complex phenotypic abnormalities including short stature, learning difficulties, and neoplasia. Moreover, memory loss associated with RTS is specifically due to lack of CBP HAT activity which can be reversed by treatment with specific histone deacetylase inhibitors (HDACs), indicative of a critical role for appropriate histone acetylation in long-term potentiation, learning, and memory. Consistent with these studies, this study provides evidence supporting a role for Tip60 HAT activity in regulating neuronal gene expression profiles required for nervous system function. This study has shown that dTip60 protein is robustly produced in the embryonic nervous system, is localized in the nuclei of brain and CNS cells, and that depletion of Tip60 HAT activity in these tissues results in fly lethality. Importantly, gene ontology (GO) analysis shows good correlation with these dTip60 protein localization studies in that a substantial number of dTip60 HAT dependent target genes are enriched for neuronal related processes, with 17 clusters linked to diverse nervous system processes and one cluster linked to muscle development. Intriguingly, these were the only tissue-specific related processes identified in microarray analysis, although some cell-specific processes may have been diluted out due to the mixed whole larvae sample preparations used for analysis. A role for dTip60 in neuronal specific function is not unprecedented, with a previous study identifying the dTIP60 gene through its accession number as a potential novel neural precursor gene in a Drosophila differential embryonic head cDNA screen (Brody, 2002), although its identity at the time remained uncharacterized. Moreover, preferential expression of TIP60 in the mouse brain has been reported and a recent study reported Bap55 as a chromatin remodeling factor that functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting in Drosophila (Tea, 2011). Taken together, these results demonstrate yet another example of the importance of HAT function during neurogenesis, and add dTip60 to the growing list of HAT chromatin regulators critical for nervous system function (Lorbeck, 2011).

Recent studies support an emerging hypothesis that inappropriate changes of specific acetylation marks in chromatin in the adult brain lead to gene misregulation that drives cognitive decline and specifically, memory impairment. These studies demonstrate that in learning assays, aged mice show a specific deregulation of histone H4 lysine 12 (H4K12) acetylation that corresponds with the misregulation of hippocampal gene expression profiles associated with learning and memory. Importantly, these effects can be reversed by restoring physiological levels of H4K12 acetylation. Thus, it is postulated that as individuals age, the accumulation of inappropriate changes in H4K12 acetylation, as well as additional acetylation and methylation marks, lead to altered transcription of neurogenic genes with subsequent negative consequences on cognitive function. Although the HAT activity of CBP has been implicated in learning and memory linked gene regulation, additional specific HATs important in these processes remain to be identified. This study shows that Tip60 protein is produced robustly in specific cells of the brain and CNS, and that Tip60 HAT activity is essential for appropriate levels of endogenous histone H4 acetylation, in vivo. Moreover, it was shown that Tip60 is essential for brain and CNS development, and intriguingly, is linked to the regulation of certain neuronal genes associated with various forms of behavior, learning, memory and synaptic function processes. Based on these results, it is tempting to speculate that Tip60 HAT activity may be involved in marking CNS chromatin important for learning and memory-linked gene regulation. Consistent with this concept, Tip60 HAT activity has been implicated in the age-related neurodegenerative disorder Alzheimer's disease (AD) via its HAT dependent complex formation with the C-terminal fragment of the amyloid precursor protein (AICD-APP) and linker protein Fe65. Recruitment of this complex is critical for the epigenetic regulation of certain genes linked to AD progression. Future investigation into the molecular mechanisms underlying Tip60 HAT function in specific neuronal processes in the fly, particularly those associated with learning and memory, should enhance understanding into the link between acetylation, cognitive aging and age-related neurodegenerative disorders (Lorbeck, 2011).

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. This study used the Drosophila neuromuscular junction (NMJ) as a well-characterized synapse model to identify HATs that control synaptic remodeling and structure. 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, alpha-tubulin acetylation levels of microtubules specifically extending into the terminal synaptic boutons are reduced in response to dTip60 HAT reduction. These 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 implications for dTip60 HAT dependant epigenetic mechanisms underlying cognitive function (Sarthi, 2011).

This report investigated a role for dTip60 in synapse development and function using the highly characterized Drosophila neuromuscular junction as a model system. dTip60 is highly concentrated at larval motor neuron synaptic boutons and is localized both pre and post synaptically, suggesting that dTip60 plays a role on both pre and postsynaptic sides of the NMJ in the differentiation of these neurons. In support of this possibility, it was shown that presynaptic depletion of dTip60 in the nervous system using GAL4 inducible fly lines dTip60RNAi and HAT defective dTip60E421Q results in a significant expansion of synaptic bouton number, indicating that this HAT negatively controls synaptic bouton formation and differentiation at the presynaptic side of the NMJ during the third instar larval stage. Interestingly, only type Is and not type Ib bouton numbers undergo significant expansion in response to dTip60 loss, supporting partial specificity in dTip60 function in certain bouton types. In addition to an increase in type Is boutons, an increase was observed in the production of satellite boutons in response to dTip60 loss. Satellite bouton production has been observed for certain Drosophila proteins that affect synaptic plasticity, including overexpression of the Drosophila amyloid precursor protein APPL, a pan-neuronal protein implicated in Alzheimer's disease and Shaggy, the Drosophila homolog of the glycogen synthase kinase 3 (GSK3β) a kinase that negatively controls NMJ growth via microtubule cytoskeleton dynamics. It is thought that satellite bouton formation is caused by an increased rate of sprouting and subsequent abnormal bouton differentiation, thus implicating dTip60 in these processes. Remarkably, this study found that there is an opposite effect on synaptic bouton number at the NMJ in response to postsynaptic Tip60 knockdown using both dTip60RNAi and dTip60E431Q HAT defective lines when compared to presynaptic knockdown, in that there is a significant reduction in bouton number, with virtually no formation of satellite boutons. Consistent with this finding, there are a number of NMJ proteins that contribute both pre and post-synaptically in the control of synaptic plasticity. For example, Discs Large (Dlg) is localized to type-1 glutamatergic synaptic terminals pre and post synaptically where it serves as a major scaffolding component at the larval NMJ. Spastin is another protein expressed at the NMJ that is concentrated both pre and post-synaptically with inappropriate localization having effects on microtubule stability that affect synaptic growth. The translational repressor Nanos also influences bouton number at the Drosophila NMJ from both the pre and postsynaptic sides. Importantly, anterograde and retrograde modes of signaling are required at sites of synaptic contact and these signaling pathways play critical roles in the formation, differentiation and plasticity of synaptic connections. For example, synaptic development at the Drosophila NMJ is dependent upon the bidirectional influence of wingless signaling on both pre and postsynaptic structures via distinct intracellular pathways. Moreover, specific pre and postsynaptic levels of the cell adhesion molecule Fasciclin II are required to regulate appropriate synaptic growth via signaling through the fly homolog of amyloid precursor protein APPL. Intriguingly, dTip60 has also been shown to be involved in APP signaling pathways via its complex formation with the C-terminal fragment of the amyloid precursor protein (APP) known as the APP intracellular domain (AICD) and linker protein Fe65 (Baek, 2000, Cao, 2001, von Rotz, 2004; Schettini, 2010). Thus, based on the pre and postsynaptic staining pattern of dTip60, and the opposite phenotypic consequences on bouton number that results from its pre versus postsynaptic reduction, it is tempting to speculate that dTip60 may also be required for bi-directional signaling at the NMJ to regulate synapse formation and function (Sarthi, 2011).

Active zones are presynaptic specializations where synaptic vesicles accumulate and fuse to the plasma membrane in response to an action potential. Importantly, they are located in perfect opposition to the glutamate receptors on the postsynaptic side of the NMJ, and thus are a commonly used marker for both synapse number and functionality in synaptic transmission. Electron-dense cytoplasmic projections termed T-bars are often observed that extend from the active zone into the presynaptic cytoplasm and are believed to facilitate vesicle movement to this site to mediate neurotransmitter release. Bruchpilot (Brp) is a scaffolding protein found in Drosophila at the active zones where it localizes to T-bars and is essential for synaptic transmission. This study shows that although there was an increase in bouton number that resulted from presynaptic dTip60 loss, the boutons show a significant reduction in the abundance in the active zone synaptic function marker Bruchpilot, suggesting a decrease in their neurotransmitter function and linking dTip60 HAT activity to this process. As postsynaptic reduction of dTip60 results in a significant reduction of 1 s boutons at the NMJ, it will be important for future studies to investigate whether these remaining boutons are also negatively impacted, in order to determine whether there is postsynaptic contribution of dTip60 on Bruchpilot expression and bouton function. Intriguingly, the precise regulation of Brp mediated neurotransmission process has been shown to be critical for higher order nervous system function that includes learning, memory and cognition. Thus, it is tempting to speculate that dTip60 HAT activity plays a critical role in the control of neurotransmission important for these processes (Sarthi, 2011).

Tip60 HAT activity has been implicated in the age-related neurodegenerative disorder Alzheimer's disease (AD) via its HAT dependent complex formation with the C-terminal fragment of the amyloid precursor protein (APP) known as the APP intracellular domain (AICD) and linker protein Fe65. The association of these proteins, termed the AFT complex, epigenetically control the transcriptional regulation of target genes involved in neuronal function. Interestingly, the Drosophila NMJ phenotype that results from presynaptic inhibition of Shaggy kinase activity is very similar to the dTip60 HAT presynaptic depletion mutant NMJ phenotype, which includes expansion of synaptic bouton number, and an increase in satellite bouton formation that is accompanied by an excess of Futsch stained microtubule loops. Shaggy is thought to function in the negative regulation of bouton expansion via phosphorylation of the MAP1B microtubule binding protein Futsch, thus inhibiting its action in promoting formation of the microtubule loops that are associated with stable bouton formation. Intriguingly, Gsk3β (the mammalian shaggy homolog) has been shown to be a direct transcriptional target of the AFT complex, where Tip60 HAT activity is required for the epigenetic control of Gsk3β transcriptional activation (von Rotz, 2004). Based on these findings, the NMJ overgrowth phenotype shared by Tip60, Shaggy and APPL (Drosophila APP homolog) mutants may suggest that these proteins are involved in overlapping transcriptional regulatory pathways that could potentially be involved in the synaptic defects observed in early AD progression. Additionally, since a decrease was observed in bruchopilot immunostaining and an increase of Futsch stained microtubule loops in dTip60 mutant larvae, it will be important in future studies to determine whether these neuronal marker genes, as well as shaggy are direct transcriptional targets of dTip60 (Sarthi, 2011).

Alternatively, dTip60 may also directly regulate synaptic microtubule architecture independent of epigenetic based transcriptional mechanisms. For example, this study found that α-tubulin acetylation levels of microtubules specifically extending into the terminal synaptic boutons are reduced in response to dominant negative HAT defective dTip60E431Q overexpression, but not dTip60RNAi induced knockdown. One possible explanation for this observation is that in the dTip60E431Q mutant, there is competition for acetylation of tubulin between the wild-type dTip60 and dTip60E431Q protein, whereas in the RNAi induced knockdown, there is still enough residual endogenous dTip60 protein available for acetylation function. Consistent with this interpretation, the decrease in tubulin acetylation levels observed in the dTip60E431Q larvae is subtle, possibly indicative of the presence of endogenous dTip60 protein and/or other endogenous factors that can still acetylate tubulin. One such endogenous factor may be the HAT Elp3, an acetylase shown to directly acetylate microtubules in cortical neurons that contributes to their migration and differentiation. These studies demonstrate that loss of Elp3 in cultured projection neuronal cells leads to severe defects in axonal branching. Intriguingly, previous studies investigating a role for Elp3 in synaptic plasticity demonstrate a very similar phenotype to dTip60 mutants, in that bouton expansion is significantly increased in response to presynaptic RNAi induced Elp3 loss (Creppe, 2009). Thus, it will be important to determine whether expression, localization and activity of Elp3 is affected at the NMJ in dTip60 mutant lines. It is noted that the link observed between dTip60 loss and reduction of α-tubulin acetylation levels of microtubules at the NMJ is correlative in nature. Therefore, it will also be important to decipher whether dTip60 acts indirectly or directly to acetylate tubulin on specific residues and whether this acetylation directly impacts specific biological processes such as microtubule architecture and/or influences interaction with microtubule binding proteins such as Futsch. Interestingly, this study observed that although acetylation of tubulin is not significantly decreased in response to dTip60RNAi induced knockdown, pupal lethality still occurs, suggesting that the acetylation of tubulin cannot be specifically linked to nervous specific dTip60 induced lethality. These results are consistent with studies on HDAC6 knockout mice, a class II HDAC known to target K40 acetylation of α-tubulin, demonstrating that although these mice display a significant increase in tubulin acetylation in the brain, this phenomenon does not rescue disease progression in a mouse model of Huntington's disease (Sarthi, 2011).

The emerging hypothesis that age-related aberrant changes of specific acetylation marks in chromatin in the adult brain lead to gene misregulation that drives cognitive decline and specifically, memory impairment in the elderly, underscores the crucial role HATs play in cognitive ability. A role for HAT activity in learning and memory is not unprecedented, with the HAT CBP implicated as a critical component of memory consolidation via the site specific histone acetylation of chromatin in the brain that in turn, regulates long-term transcriptional changes associated with long-lasting forms of neuronal plasticity. Importantly, recent studies show that synaptic activity can influence such CBP associated histone acetylation marks, thus providing a mechanism for how external environmental stimuli can influence behavioral dependant synaptic plasticity that is linked to memory. In this model of synaptic activity-dependent epigenetic regulation, synaptic input and depolarizing stimuli cause an increase in Ca2+ intracellular levels via specific Ca2+ channels, thus activating certain kinases to phosphorylate CBP which is thought to promote its recruitment to chromatin. CBP mediated specific histone acetylation marks cooperate with additional epigenetic modifications to induce chromatin structural changes that regulate gene expression of synaptic activity-dependant genes. In support of this model, genome-wide screens of mouse cortical neurons using ChIP-seq have shown that membrane depolarization markedly enhances CBP recruitment to such enhancers. Intriguingly, neural activity can also modulate chromatin acetylation by regulating the shuttle of class II HDACs in and out of the nucleus in hippocampal neurons. As dTip60 has also been shown to shuttle between nuclear and cytoplasmic cellular compartments, with misregulation of this process associated with prostate cancer (Halkidou, 2003; Lee, 2001) it is tempting to speculate that cellular localization of Tip60 influences synaptic plasticity in a manner similar to certain HDACs. The current findings causatively linking Tip60 HAT activity to the control of synaptic bouton formation and function, in conjunction with future studies deciphering the mechanisms of how Tip60 controls such processes, should further understanding of epigenetic mechanisms underlying synaptic plasticity and memory formation in neurodevelopment, age-related cognitive decline and neurodegenerative disorders (Sarthi, 2011).

Activator-induced spread of poly(ADP-ribose) polymerase promotes nucleosome loss at Hsp70

Eukaryotic cells possess many transcriptionally regulated mechanisms to alleviate the nucleosome barrier. Dramatic changes to the chromatin structure of Drosophila melanogaster Hsp70 gene loci are dependent on the transcriptional activator, heat shock factor (HSF), and poly(ADP-ribose) polymerase (PARP). This study found that PARP is associated with the 5' end of Hsp70, and its enzymatic activity is rapidly induced by heat shock. This activation causes PARP to redistribute throughout Hsp70 loci and Poly(ADP-ribose) to concurrently accumulate in the wake of PARP's spread. HSF is necessary for both the activation of PARP's enzymatic activity and its redistribution. Upon heat shock, HSF triggers these PARP activities mechanistically by directing Tip60 acetylation of histone H2A lysine 5 at the 5′ end of Hsp70, where inactive PARP resides before heat shock. This acetylation is critical for the activation and spread of PARP as well as for the rapid nucleosome loss over the Hsp70 loci (Petesch, 2012).

This study establishes an ordered mechanism by which a transcription activator binding to a gene's regulatory region leads to rapid removal of nucleosomes throughout the gene locus. Specifically, the transcriptional activator, HSF, stimulates dTip60 acetylation of H2AK5 that in turn activates PARP, causing its redistribution along Hsp70 and reduced nucleosome occupancy over the locus. Moreover, all of these steps can be accomplished independently of transcription. This activation of PARP and its rapid spread throughout the Hsp70 HS loci demonstrate an interesting mechanism by which the nucleosome barrier can be alleviated to facilitate efficient transcription by Pol II (Petesch, 2012).

HSF and many other transcriptional activators have been classically studied for their ability to recruit or release Pol II into transcriptional elongation. The results speak to another function of HSF as an activator to direct changes in chromatin structure upon HS. HSF is able to achieve this function through physically interacting with the dTip60 complex and facilitating its recruitment to Hsp70 following HS (personal communication by Thomas Kusch to Petesch, 2012). Just as the presence of paused Pol II in non-heat-shock (NHS) conditions primes the Hsp70 gene for rapid transcriptional induction, inactive PARP bound in NHS conditions primes Hsp70 for rapid changes in chromatin structure. Interestingly, trimerization and binding of HSF to the promoter of Hsp70 precipitates the activation of both Pol II and PARP through distinct pathways that ultimately synergize to facilitate rapid and robust transcriptional activation. In vitro studies have shown that the DNA-binding and catalytic domains of PARP comprise the minimal structure sufficient for inactive PARP to bind and locally compact nucleosomes and, upon activation, release PARP from chromatin and decompact chromatin structure. Activation of PARP is known to result in the formation of linear and branched anionic polymers with upwards of 200 units of ADP-ribose. Electron micrograph structures of branched PAR make it easy to visualize how creation of these voluminous, dendritic structures causes automodified PARP to expand 3-dimensionally throughout the Hsp70 loci following HS. The results also indicate that PARP is crosslinked to Hsp70 after HS through a PAR linkage to chromatin. Although PARylation of another target, such as histones, cannot be ruled out, the results fit the simplest model where PARP is its own target. In agreement with the aforementioned in vitro studies, PARP automodification would result in its release from nucleosomes bound prior to HS, and the PAR created from this automodification could create a bridging interaction between PARP and chromatin formed during crosslinking. This also is consistent with in vivo studies showing the major target of PARylation is PARP itself. Antibodies specifically recognizing ADP-ribosylated target proteins, such as PARP or histones, are needed to identify the target of PARP following HS at Hsp70 (Petesch, 2012).

The accumulation of PAR throughout the Hsp70 locus provides additional functional insight into how activation of PARP upon HS can affect chromatin structure and transcriptional activation. PAR has remarkable chemical similarity to other nucleic acids, such as DNA and RNA, but it has twice the charge per nucleic acid residue and the potential to form nonlinear, branched structures. As such, in vitro reconstitution assays have shown that PAR has the ability to locally compete with DNA to bind histones and potentially disrupt native chromatin structure. The transient formation of PAR to alter chromatin structure followed by catabolism of PAR to return histones to its DNA template has been referred to as histone shuttling. While initially investigated to explain PARP's role in DNA damage repair, this phenomenon can be equally extended to PARP's role in facilitating transcription. Indeed, the formation of PAR at Hsp70 loci after HS results in formation of a localized compartment that aids in the local retention of transcription factors, including Pol II, to sustain continued transcription activation of Hsp70 (Zobeck, 2010). It is yet to be determined if PAR also aids in the local retention of histones that were previously measured to be lost from Hsp70 after HS (Petesch, 2012).

The activation of PARP through the acetylation of H2AK5 also ascribes a unique function to dTip60. Like PARP, Tip60 has been studied for both its roles in DNA repair and also transcriptional activation (Sapountzi, 2006). In Drosophila, dTip60 is part of a complex containing Domino, an ATPase homologous to the mammalian p400 and SRCAP proteins, which, like Swr1p in S. cerevisiae, catalyzes the exchange of histone variant H2A.Z into H2A-containing nucleosomes. Drosophila contains only one H2A variant, which has properties of both H2A.Z and the C-terminal extension of H2A.X, and, when phosphorylated, marks sites of DNA damage. Before HS, it is known that Hsp70 contains nucleosomes harboring H2Av near the 5' end of the gene that is lost upon HS. Recently, the phosphorylation of H2AvS137 was shown to globally regulate PARP activation and is necessary for full transcriptional activation of Hsp70. dTip60 acetylates K5 on H2Av that is already phosphorylated on its C-terminal domain at S137 (Kusch, 2004). This acetylation stimulates the dTip60 complex to exchange out the modified H2Av. Additionally, in vitro studies show that the ability of H4 to activate PARP is squelched in the context of a nucleosome due to H2A. Collectively, these studies suggest a model in which the phosphorylation of H2AvS137 stimulates dTip60 to acetylate H2AvK5 following its recruitment upon HS. These modifications are sufficient to stimulate the dTip60 complex to remove the modified H2Av and expose PARP to H4 and activate its enzymatic activity. The importance of H2A variant exchange has also been documented in Arabidopsis, where the Swr1 complex is also necessary for changes in chromatin structure at HS genes following HS (Petesch, 2012).

This proposed model for the order of events that lead to the activation of PARP upon HS raises many questions for future exploration. First, is the H2Av that is present before HS already phosphorylated, and what is the kinase responsible for phosphorylation? Second, is phosphorylation of H2Av necessary for dTip60 acetylation of H2AvK5 upon HS? Third, is H2AvK5Ac by itself or in combination with S137 phosphorylation sufficient for PARP activation in vitro? Fourth, is the ATPase activity of the dTip60 complex to exchange H2Av following HS necessary or sufficient for PARP activation? Finally, is the activity of PARP regulated on a genomic scale at sites with H2Av nucleosomes that are both acetylated at K5 and phosphorylated at S137 (Petesch, 2012)?

The fact that transcription-independent nucleosome loss following HS at Hsp70 is reliant on factors that respond to DNA damage provokes the question if changes in chromatin at Hsp70 are the result of a response to DNA repair. Indeed, transcriptional activation can occur in response to PARP activation from a topoisomerase II break in DNA. However, in contrast to that study, this study found that PARP is already present at Hsp70 before HS and is not recruited upon HS. Although topoisomerase II mediated breaks have been mapped to sites near the TSS of Hsp70 before HS, these breaks are not sufficient to detect active PARP at Hsp70 before HS and might be more important for the initial deposition of PARP before HS. An alternative mechanism is proposed for PARP activation whereby a transcriptional activator hijacks DNA repair proteins to aid transcriptional activation. The fact that PARP is bound near the majority of human TSSs containing Pol II as at Drosophila Hsp70, also hints at the generality for a mechanism whereby activation of prebound PARP leads to changes in chromatin structure and ultimately contributes to gene expression (Petesch, 2012).

The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting

The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. This study found that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (TIP60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. These results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions (Tea, 2011).

The stereotyped organization of the Drosophila olfactory system makes it an attractive model to study wiring specificity. The first olfactory processing center is the antennal lobe, a bilaterally symmetric structure at the anterior of the Drosophila brain. It is composed of approximately 50 glomeruli in a three-dimensional organization. Each olfactory projection neuron (PN) targets its dendrites to one of those glomeruli to make synaptic connections with a specific class of olfactory receptor neurons. Each PN sends its axon stereotypically to higher brain centers (Tea, 2011).

During development, the dendrites of PNs pattern the antennal lobe prior to axons of olfactory receptor neurons. The specificity of PN dendrite targeting is largely genetically pre-determined by the cell-autonomous action of transcription factors, several of which have been previously described. Furthermore, chromatin remodeling factors have been shown to play an important role in PN wiring (Tea, 2010), although very little is currently known about their specific functions. This study reports a genetic screen for additional factors that regulate PN dendrite wiring specificity; Brahma associated protein 55 kD (Bap55) was identified as a regulator of PN dendrite wiring specificity as part of the TIP60 chromatin remodeling complex (Tea, 2011).

Bap55 is an actin-related protein, the majority of which physically associates with the Brahma (BRM) chromatin remodeling complex in Drosophila embryo extracts. There are two distinct BRM complexes: BAP (Brahma associated proteins; homologous to yeast SWI/SNF) and PBAP (Polybromo-associated BAP; homologous to yeast RSC), both of which contain Brahma, Bap55, and Snf5-Related 1 (Snr1). The human homologs of the BAP and PBAP complexes are called the BAF (Brg1 associated factors) and PBAF (Polybromo-associated BAF) complexes, respectively. The BRM/BAF complexes are members of the SWI/SNF family of ATP-dependent chromatin-remodeling complexes, and have been shown to both activate and repress gene transcription, in some cases, of the same gene (Tea, 2011).

In experiments purifying proteins in complex with tagged Drosophila Pontin in S2 cells, Bap55 was also co-purified as a part of the TIP60 complex, as determined by mass spectrometry. The TIP60 histone acetyltransferase complex has been shown to be involved in many processes, including both transcriptional activation and repression. The complex contains many components, including Bap55, Domino (Dom), and Enhancer of Polycomb (E(Pc)). Dom, homologous to human p400, is the catalytic DNA-dependent ATPase; its ATPase domain is highly similar to Drosophila Brahma and human BRG1 ATPase domains. E(Pc) is homologous to human EPC1 and EPC2 and is an unusual member of the Polycomb group; it does not exhibit homeotic transformations on its own, but rather enhances mutations in other Polycomb group genes (Tea, 2011).

This study provides evidence that Bap55 functions as a part of the TIP60 complex rather than the BRM complex in postmitotic PNs to control their dendrite wiring specificity (Tea, 2011).

To further understanding of dendrite wiring specificity in Drosophila olfactory PNs, a MARCM-based forward genetic screen was performed using piggyBac insertional mutants. MARCM allows visualization and genetic manipulation of single cell or neuroblast clones in an otherwise heterozygous background, permitting the study of essential genes in mosaic animals. In this screen, GH146-GAL4 was used to label a single PN born soon after larval hatching, which in wild-type (WT) animals always projects its dendrites to the dorsolateral glomerulus DL1 in the posterior of the antennal lobe. The DL1 PN also exhibits a stereotyped axon projection, forming an L-shaped pattern in the lateral horn, with additional branches in the mushroom body calyx. A mutant, called LL05955, was identified in which DL1 PNs mistargeted to the dorsolateral glomerulus DA4l in the anterior of the antennal lobe. This phenotype is strikingly specific, with 100% penetrance. Arborization of mutant axons, however, was not obviously altered. The piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. LL05955 is inserted into the coding sequence of Bap55, encoding a homolog of human BAF53a and BAF53b. Precise excision of the piggyBac insertion reverted the dendrite mistargeting phenotype, confirming that disruption of the Bap55 gene causes the dendrite mistargeting (Tea, 2011).

In addition to causing DL1 mistargeting, Bap55 mutants also display neuroblast clone phenotypes. In WT, GH146-GAL4 can label three distinct types of PN neuroblast clones generated in newly hatched larvae. Two of these clones, the anterodorsal neuroblast clone and the lateral neuroblast clone, possess cell bodies that lie dorsal or lateral to the antennal lobe, respectively. PNs from these two lineages project their dendrites to stereotyped and nonoverlapping subsets of glomeruli in the antennal lobe. The third type of clone, the ventral neuroblast clone, has cell bodies that lie ventral to the antennal lobe and dendrites that target throughout the antennal lobe due to the inclusion of at least one PN that elaborates its dendrites to all glomeruli (Tea, 2011).

In Bap55-/- PNs, anterodorsal neuroblast clones display a mild reduction in cell number, and their dendrites are abnormally clustered in the anterior dorsal region of the antennal lobe, including the DA4l glomerulus. Lateral neuroblast clones display a severe reduction in cell number, and the remaining dendrites are unable to target to many glomeruli throughout the antennal lobe. Ventral neuroblast clones display a mild reduction in cell number and a reduced dendrite mass throughout the antennal lobe. During development, the lateral neuroblast first gives rise to local interneurons before switching to produce PNs; in mutants affecting cell proliferation, this property of the lateral neuroblast displays as a severe reduction in GH146-labeled PNs. The severely reduced cell number in Bap55 mutants suggests that Bap55 is essential for neuroblast proliferation or neuronal survival. In the anterodorsal and ventral neuroblasts, PN numbers are not drastically changed; thus, the phenotypes indicate that Bap55 is important for dendrite targeting in multiple classes of PNs (Tea, 2011).

In WT, Mz19-GAL4 labels a subset of the GH146-GAL4 labeling pattern. It labels a small number of PNs derived from two neuroblasts, which can be clearly identified in WT clones generated in newly hatched larvae. Anterodorsal neuroblast clones target their dendrites to the VA1d glomerulus, as well as the DC3 glomerulus residing immediately posterior to VA1d (not easily visible in confocal stacks). Lateral neuroblast clones target all dendrites to the DA1 glomerulus. Unlike GH146-GAL4, WT Mz19-GAL4 never labels ventral neuroblast clones because it is not normally expressed in those cells (Tea, 2011).

In Bap55 mutant PN clones, however, Mz19-GAL4 labels additional PNs in anterodorsal, lateral, and ventral clones compared to their WT counterparts. This phenotype suggests that some Mz19-negative PNs now express Mz19-GAL4. In anterodorsal clones, Mz19-GAL4 labels additional cells, although not as many as are labeled by GH146-GAL4. The PNs also mistarget their dendrites to the anterior antennal lobe, similar to mutant GH146-GAL4 anterodorsal neuroblast clones. WT lateral neuroblast clones normally contain GH146-positive PNs and GH146-negative local interneurons. In Bap55-/- lateral neuroblast clones, Mz19-GAL4 predominantly labels local interneurons that send their processes to many glomeruli throughout the antennal lobe and do not send axon projections to higher brain centers. Lateral clones also show ectopic PN labeling with a lower frequency. The Bap55 mutant appears to cause derepression of Mz19-GAL4, resulting in labeled local interneurons. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in Bap55 mutants. This further indicates a derepression of the Mz19-GAL4 labeling pattern (Tea, 2011).

Unlike GH146-GAL4, WT Mz19-GAL4 never labels single cell clones when clone induction is performed in newly hatched larvae. This is because Mz19-GAL4 is not expressed in the DL1 PN, the only GH146-positive cell generated during this heat shock time of clone generation. However, in Bap55 mutant PN clones, Mz19-GAL4 ectopically labels single cell anterodorsal PN clones targeting to the DA4l glomerulus, which show an L-shaped pattern in the lateral horn with branches in the mushroom body calyx, similar to GH146-GAL4 labeling. The simplest interpretation is that this compound phenotype reflects first a derepression of Mz19-GAL4 in the DL1 PN, and second a DL1 to DA4l mistargeting phenotype in Bap55 mutants (Tea, 2011).

To test whether Bap55 functions in the neuroblast or postmitotically in PNs, GH146-GAL4, which expresses only in postmitotic PNs, was used to express UAS-Bap55 in a Bap55-/- single cell clone. The dendrite mistargeting phenotype was shown to be rescued to the WT DL1 glomerulus and it is concluded that Bap55 functions postmitotically to regulate PN dendrite targeting. The axon phenotype remains the stereotypical L-shaped pattern (Tea, 2011).

The Drosophila Bap55 protein is 70% similar and 54% identical to human BAF53a and 71% similar and 55% identical to human BAF53b. BAF53a and b are 91% similar and 84% identical to each other. Using GH146-GAL4 to express human BAF53a or b in a Bap55-/- single cell clone, it was found that the human homologs can effectively rescue the Bap55-/- dendrite mistargeting phenotype. Interestingly, both also cause the de novo DM6 dendrite and ventral axon mistargeting phenotypes in 6 out of 19 cases for BAF53a and 2 out of 32 cases for BAF53b. Thus, human BAF53a and b can largely replace the function of Drosophila Bap55 in PNs (Tea, 2011).

To address whether Bap55 functions as a part of the BRM complex in PN dendrite targeting, two additional BRM complex mutants were tested for their PN dendrite phenotypes. First, Brahma (brm), the catalytic ATPase subunit of the BRM complex, which is required for the activation of many homeotic genes in Drosophila, was tested. Null mutations have been shown to decrease cell viability and cause peripheral nervous system defects. RNA interference knockdown of brm in embryonic class I da neurons revealed dendrite misrouting phenotypes, although not identical to the Bap55 phenotype. The human homologs of brm, BRM and BRG1 (Brahma-related gene-1), both have DNA-dependent ATPase activity. Inactivation of BRM in mice does not yield obvious neural phenotypes, but inactivation of BRG1 in neural progenitors results in reduced proliferation. BRG1 is likely to be required for various aspects of neural development, including proper neural tube development (Tea, 2011).

In PNs, brm mutants displayed anterodorsal single cell clone mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone differing from the next. This is in contrast to the highly stereotyped DA4l mistargeting of Bap55 mutants. brm-/- neuroblast clones also displayed phenotypes where dendrites make small, meandering projections throughout the antennal lobe, which does not resemble the Bap55-/- phenotype. They additionally exhibit a perturbed cell morphology phenotype, which is markedly more severe than the Bap55 mutant phenotype (Tea, 2011).

Next, Snr1, a highly conserved subunit of the BRM complex, was tested. It is required to restrict BRM complex activity during the development of wing vein and intervein cells and functions as a growth regulator. Its human homolog, SNF5, is strongly correlated with many cancers, yet little is known about its specific role in neurons (Tea, 2011).

In PNs, Snr1 mutants displayed similar phenotypes to brm mutants. The single cell clones displayed mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone demonstrating a unique phenotype. The neuroblast clones exhibited small meandering dendrites throughout the antennal lobe, which also showed extremely perturbed cell morphology. These results, especially the non-sterotyped single cell clone phenotypes, indicate that the BRM complex functions differently from Bap55 in controlling PN dendrite targeting (Tea, 2011).

brm and Snr1 mutants were further analyzed with Mz19-GAL4 to determine whether their phenotypes resembled the Bap55 mutant phenotype of derepression. It was not possible to generate any labeled clones, indicating one of three possibilities: increased cell death, severe cell proliferation defects, or repression of the Mz19-GAL4 labeling pattern. In any of the three cases, the phenotype does not resemble the Bap55-/- mutant phenotype of abnormal activation of Mz19-GAL4 in single cell or neuroblast clones, indicating that the BRM complex functions differently from Bap55 in PNs (Tea, 2011).

In the same screen in which the Bap55 mutation was identified, LL05537, a mutation in dom that resulted in a qualitatively similar phenotype to Bap55 mutants was identified. dom-/- DL1 PNs split their dendrites between the posterior glomerulus DL1 and the anterior glomerulus DA4l. Their axons exhibit a WT L-shaped pattern in the lateral horn (Tea, 2011).

The LL05537 allele contains a piggyBac insertion in an intron just upstream of the translation start of dom. Because the piggyBac insertion contains splice acceptor sites and stop codons in all three coding frames, this allele likely disrupts all isoforms of dom. Similarly to Bap55, the piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. Precise excision of the piggyBac insertion reverted the dendrite targeting phenotype, confirming that disruption of the dom gene causes the dendrite mistargeting. In addition, a BAC transgene that contains the entire dom transcriptional unit rescued the dom-/- mutant phenotypes (Tea, 2011).

Dom is the catalytic DNA-dependent ATPase of the TIP60 complex and has been shown to contribute to a repressive chromatin structure and silencing of homeotic genes. Dom is a member of the SWI/SNF family and its ATPase domain is highly similar to the Drosophila Brahma and human BRG1 ATPase domains. The human homolog of Dom is p400, which is important for regulating nucleosome stability during repair of double-stranded DNA breaks and an important regulator of embryonic stem cell identity (Tea, 2011).

To determine whether Bap55 and Dom genetically interact, UAS-Bap55 was expressed in a dom-/- PN. This manipulation did not significantly alter the dendrite phenotype. The axon branching pattern also was not altered (Tea, 2011).

Another component of the TIP60 complex, E(Pc), was also examined. In Drosophila, E(Pc) is a suppressor of position-effect variegation and heterozygous mutations in E(Pc) result in an increase in homologous recombination over nonhomologous end joining at double-stranded DNA breaks. Following ionizing radiation, heterozygous animals also exhibit higher genome stability and lower incidence of apoptosis. Yet little is known about its role in neurons (Tea, 2011).

In this study, it was found that E(Pc)-/- DL1 PN dendrites also mistarget to the anterior glomerulus DA4l and exhibit the stereotyped L-shaped axon pattern in the lateral horn. A BAC transgene that contains the entire E(Pc) transcription unit rescued the E(Pc) mutant phenotypes. To determine whether Bap55 and E(Pc) genetically interact, UAS-Bap55 was expressed in an E(Pc)-/- DL1 PN. This manipulation caused the dendrites to split between the DA4l and DM6 glomeruli, and resulted in axons targeting ventrally to the lateral horn (Tea, 2011).

Neuroblast clones mutant for dom also exhibit dendrite mistargeting phenotypes to inappropriate glomeruli throughout the antennal lobe. Anterodorsal and lateral neuroblast clones show a very mild reduction in cell number and their dendrites do not target to the full set of proper glomeruli. Ventral neuroblast clones, when compared to WT, exhibit incomplete targeting throughout the antennal lobe (Tea, 2011).

Further analysis of dom mutants by labeling with Mz19-GAL4 revealed the same derepression as in Bap55 mutants. dom mutant Mz19-GAL4 PN clones also label anterodorsal, lateral, and ventral neuroblast clones with phenotypes similar to GH146-GAL4 labeled neuroblast clones. In anterodorsal and lateral neuroblast clones, Mz19-GAL4 labels a large number of PNs that target to many glomeruli throughout the antennal lobe, although the cell number is smaller than GH146-GAL4 labeling. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in dom mutants. Mz19-GAL4 also labels single cell clones that split their dendrites between the DA4l and DL1 glomeruli and form the stereotypical L-shaped axon pattern in the lateral horn. As in Bap55 mutants, this compound phenotype likely results from ectopic labeling of a DL1 PN, which further mistargets to DA4l (Tea, 2011).

The E(Pc) phenotypes in GH146 and Mz19-GAL4 labeled neuroblast clones, as well as Mz19-GAL4 labeled single cell clones, displayed similar phenotypes to dom as described above. The phenotypic similarities in single cell clone dendrite mistargeting and derepression of a PN-GAL4 in mutations that disrupt Bap55, dom and E(Pc) strongly suggest that these three proteins act together in the TIP60 complex to regulate PN development (Tea, 2011).

This study has demonstrated a similar role for three members of the TIP60 complex in olfactory PN wiring. The TIP60 complex plays a very specific role in controlling dendrite wiring specificity, with a precise mistargeting of the dendrite mass in Bap55, dom, and E(Pc) mutants. This specific DL1 to DA4l mistargeting phenotype has only been seen in these three mutants, out of approximately 4,000 other insertional and EMS mutants screened, supporting the conclusion that the TIP60 complex has a specific function in controlling PN dendrite targeting. TIP60 complex mutants show discrete glomerular mistargeting, rather than randomly distributed dendrite spillover to different glomeruli. In contrast, perturbation of individual cell surface receptors often leads to variable mistargeted dendrites that do not necessarily obey glomerular borders, possibly reflecting the combinatorial use of many cell surface effector molecules. Even transcription factor mutants yield variable phenotypes. Interestingly, BRM complex mutants yield non-stereotyped phenotypes in PNs. No stereotyped glomerular targeting was seen for brm or Snr1 mutant dendrites; each PN spreads its dendrites across different glomeruli. These data suggest that different chromatin remodeling complexes play distinct roles in regulating neuronal differentiation. The uni- or bi-glomerular targeting to specific glomeruli implies that the TIP60 complex sits at the top of a regulatory hierarchy to orchestrate an entire transcriptional program of regulation (Tea, 2011).

This study suggests a function for Bap55 in Drosophila olfactory PN development as a part of the TIP60 complex rather than the BRM complex. Another possibility could be that Bap55 also serves as the interface between the BRM and TIP60 complexes. While loss of core BRM complex components results in a more general defect, loss of Bap55 could specifically disrupt interactions with the TIP60 complex but maintain other BRM complex functions, causing a more specific targeting phenotype mimicking loss of TIP60 complex components (Tea, 2011).

Interestingly, both human BAF53a and b can significantly rescue the Bap55-/- phenotype. Though in mammals BAF53a is expressed in neural progenitors and BAF53b is expressed in postmitotic neurons, they can perform the same postmitotic function in Drosophila PNs. Further, both can function with the TIP60 complex in PNs to regulate wiring specificity. These data suggest that the functions for BAF53a and b (in neural precursors and postmitotic neurons, respectively) diverge after the evolutionary split between vertebrates and insects (Tea, 2011).

The discrete glomerular states of the mistargeting phenotypes may suggest a role for the TIP60 complex upstream of a regulatory hierarchy determining PN targeting decisions. It is possible that disrupting various components changes the composition of the complex. Additionally, overexpression of Bap55 in various mutant backgrounds might alter the sensitive stoichiometry of the TIP60 complex, resulting in targeting to different but still distinct glomeruli (Tea, 2011).

Several mutants have been identified that cause DL1 PNs to mistarget to areas near the DM6 glomerulus (Tea, 2010). Interestingly, WT DM6 PNs have the most similar lateral horn axon arborization pattern to DL1 PNs. It is hypothesized that the transcriptional code for DM6 is similar to that of DL1, which is at least partially regulated by the TIP60 complex. The genes described in this manuscript are the only mutants that have yielded specific DA4l mistargeting to date. It is possible that the targeting 'code' for DA4l, DL1, and DM6 may be most similar, such that perturbation of the TIP60 complex might result in reprogramming of dendrite targeting. PNs have previously been shown to be pre-specified by birth order. Yet DA4l is born in early embryogenesis, DL1 is born in early larva, and DM6 is born in late larva. This implies that the TIP60 transcriptional code does not correlate with PN birth order. The mechanisms by which the TIP60 complex specifies PN dendrite targeting remain to be determined (Tea, 2011).

This study has characterize PN phenotypes of mutants in the BRM and TIP60 complexes, with a focus on Bap55, which is shared by the two complexes. The TIP60 complex was found to play a very specific role in regulating PN dendrite targeting; mutants mistarget from the DL1 to the DA4l glomerulus. This specific mistargeting phenotype suggests that TIP60 controls a transcriptional program important for making dendrite targeting decisions (Tea, 2011).

Epigenetic control of learning and memory in Drosophila by Tip60 HAT action

Disruption of epigenetic gene control mechanisms in the brain causes significant cognitive impairment that is a debilitating hallmark of most neurodegenerative disorders including Alzheimer's disease (AD). Histone acetylation is one of the best characterized of these epigenetic mechanisms that is critical for regulating learning and memory associated gene expression profiles, yet the specific histone acetyltransferases (HATs) that mediate these effects have yet to be fully characterized. This study investigated an epigenetic role for the HAT Tip60 in learning and memory formation using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. Tip60 is endogenously expressed in the Kenyon cells, the intrinsic neurons of the MB and in the MB axonal lobes. Targeted loss of Tip60 HAT activity in the MB causes thinner and shorter axonal lobes while increasing Tip60 HAT levels cause no morphological defects. Functional consequences of both loss and gain of Tip60 HAT levels in the MB are evidenced by defects in immediate recall memory. ChIP-Seq analysis reveals that Tip60 target genes are enriched for functions in cognitive processes and accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, it was found that both learning and immediate recall memory deficits that occur under AD associated amyloid precursor protein (APP) induced neurodegenerative conditions can be effectively rescued by increasing Tip60 HAT levels specifically in the MB. Together, these findings uncover an epigenetic transcriptional regulatory role for Tip60 in cognitive function and highlight the potential of HAT activators as a therapeutic option for neurodegenerative disorders (Xu, 2014).

Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin

Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex, which includes Domino as a subunit. In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. Genetic interactions have been demonstrated between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. It is concluded that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin (Qi, 2006).

A fundamental regulatory step in transcription and other DNA-dependent processes in eukaryotes is the control of chromatin structure, which regulates access of proteins to DNA. Histone acetylation and the protein complexes that mediate this modification have been linked to activation of transcription. It is believed that lysine acetylation of histone N termini results in less compact chromatin by neutralizing the positive charge of histones and that the acetyl groups are recognized by regulatory proteins that promote transcription. However, it is becoming clear that histone acetyltransferases (HATs) can have functions other than facilitating transcription. For example, the TIP60 HAT complex has been implicated in DNA repair in yeast, flies, and mammals. This study investigated the role of Drosophila Reptin and other TIP60 components in chromatin regulation in vivo (Qi, 2006).

The Reptin protein, also known as TIP48, TIP49b, or RUVBL2, is related to bacterial RuvB, an ATP-dependent DNA helicase that promotes branch migration in Holliday junctions. Reptin, and the related Pontin (TIP49, TIP49a, or RUVBL1) protein, possess intrinsic ATPase and helicase activities and can heterodimerize. In yeast, both Reptin and Pontin are part of the INO80 chromatin-remodeling complex, as well as the Swr1 complex that can exchange histone H2A with the variant histone H2A.Z. Reptin and Pontin appear to play antagonistic roles in development by regulating Wnt signaling and heart growth in zebrafish embryos. Mammalian Reptin and Pontin are present in TIP60 HAT complexes, which are involved in induction of apoptosis in response to DNA damage and which interact with the c-Myc protein to promote its oncogenic activity (Qi, 2006 and references therein).

TIP60 is a HAT of the MYST family (Utley, 2003). The homologous yeast protein Esa1 is the catalytic subunit of the nucleosome acetyltransferase of H4 (NuA4) complex, which acetylates lysines in histone H4 and H2A (Doyon, 2004). In Drosophila, the TIP60 complex acetylates the phosphorylated variant histone H2Av after DNA double-strand breaks and exchanges it with unmodified H2Av. The composition of TIP60 and NuA4 complexes has recently been determined. TIP60 (yeast Esa1), ING3 (Yng2), and Enhancer of Polycomb (EPC1, yeast Epl1) form a core complex that is sufficient for acetylation of histones in nucleosomes. Mammalian and Drosophila TIP60 complexes contain four subunits not present in yeast NuA4: Brd8, Reptin, Pontin, and Domino (also known as p400), the homolog of yeast Swr1 (Qi, 2006).

Polycomb group (PcG) proteins are evolutionarily conserved chromatin regulators that maintain appropriate expression patterns of developmental control genes, such as the Hox genes. PcG proteins are generally repressors that maintain the off state of genes and exist in at least two distinct protein complexes. The Esc-E(z) complex is a histone methyltransferase that includes the catalytic subunit Enhancer of zeste [E(z)], as well as the Extra sex combs (Esc) and Suppressor of zeste 12 [Su(z)12] subunits. Another complex purified from Drosophila embryos, Polycomb repressive complex 1 (PRC1) has a mass of >1 MDa. In addition to genetically identified PcG proteins, it includes TFIID subunits, the Reptin protein, and other polypeptides. The PRC1 complex can block chromatin remodeling by the SWI/SNF complex in vitro. A core PRC1 complex consisting of Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and dRING1/Sex combs extra (Sce) is sufficient for the in vitro activities of PRC1. Recently, it was shown that dRing1/Sce as well as its mammalian orthologs are E3 ubiquitin ligases that monoubiquitylate histone H2A (Qi, 2006 and references therein).

This study investigates the role of Drosophila Reptin in chromatin regulation. Reptin is shown to interact genetically with PcG gene products and suppresses position-effect variegation (PEV), properties shared by other Drosophila TIP60 complex components. It is suggested that the fly TIP60 complex regulates epigenetic processes leading to a repressive chromatin state. This is a novel activity of a HAT complex that has previously been implicated in transcription activation and DNA repair (Qi, 2006).

It is proposed that Reptin acts as a subunit of the TIP60 HAT complex to generate a repressive chromatin state. This is a novel activity of a HAT complex previously shown to promote transcription. This study shows that Reptin copurifes with the Polycomb complex PRC1. This prompted an investigation of whether the biochemical interaction with PRC1 was accompanied by a genetic interaction. It was shown that Reptin and PRC1 components genetically interact to regulate expression of the Hox gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl. Although no interactions were detected between reptin heterozygous mutants and several PREs tested, a PRE from the Ubx gene is derepressed in reptin homozygous mutant cells. This shows that Reptin contributes an essential function to the activity of this PRE. However, unlike most PcG genes, reptin homozygous mutants do not derepress endogenous Hox gene expression. It appears that repression of endogenous Hox genes is more complex and not as sensitive to the loss of Reptin as the Ubx PRE. In contrast to most PcG genes, reptin mutants suppress PEV. Interestingly, derepression of the Ubx PRE also occurs in embryos mutant for other suppressors of PEV, indicating that this PRE may be highly sensitive to the chromatin environment in its vicinity. Since reptin mutants suppress PEV and fail to derepress endogenous Hox gene expression, reptin is not considered a bona fide PcG gene, and it is found unlikely that Reptin protein contributes an essential function to the PRC1 complex. In fact, the biochemical activities ascribed to PRC1 can be reconstituted either with recombinant dRing1/Sce or with four core components whose activity can be further enhanced by the DNA-binding proteins Zeste and GAGA (Qi, 2006).

Given that Reptin is present in TIP60 complexes in mammals and recently was shown to be a component of a Drosophila TIP60 complex, the possibility is considered that the genetic interactions observed with PcG genes are due to the presence of Reptin in the fly TIP60 complex. The products of two previously characterized Drosophila genes, E(Pc) and domino, are also present in the TIP60 complex. Strikingly, E(Pc) and domino mutants share with reptin the ability to genetically interact with PcG genes and suppress PEV. E(Pc) is an unusual PcG gene that has very minor effects on Hox gene expression, and unlike most PcG genes, modifies PEV. In both yeast and humans, E(Pc) homologs form a core complex with Esa1 (TIP60) and Yng2 (ING3) that is sufficient for the nucleosomal acetylation of histones H4 and H2A by the NuA4 complex (Boudreault, 2003; Doyon, 2004). That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests that Reptin functions through the fly TIP60 complex (Qi, 2006).

Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast. Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes. Intriguingly, an involvement of Htz1 (H2A.Z) in controlling the spreading of silenced chromatin has recently been demonstrated in yeast. Exchange of variant histones may be a conserved feature of chromatin regulation since a recent report demonstrates that Drosophila H2Av behaves genetically as a PcG gene and suppresses PEV. Domino exchanges phosphorylated and acetylated H2Av for unmodified H2Av after DNA damage (Kusch, 2004). However, no change was found in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (Qi, 2006).

A P-element insertion was identified in the gene encoding one additional TIP60 complex component, the chromodomain-containing protein MRG15. Human MRG15 (MORF-related gene on chromosome 15) has been implicated in cellular senescence and regulation of the B-myb promoter. Both human and yeast (Eaf3/Alp13) MRG15 have been found in Sin3/HDAC complexes in addition to the TIP60 (NuA4) complex, where it directs the histone deacetylase to coding regions through interaction of its chromodomain with methylated histone H3 lysine 36. This study found that MRG15 mutant flies interact with PcG genes and suppress PEV, just as other TIP60 complex components do. This is taken as further support of the conclusion that Reptin's effects on chromatin processes are mediated through its association with the fly TIP60 complex (Qi, 2006).

What is the basis for the genetic interaction between TIP60 components and PcG genes? One possibility is that the TIP60 complex regulates PcG expression. However, no reduction was observed in Pc expression in reptin mutant embryos. Another possibility is that the enzymatic activities of the TIP60 complex cooperate with PcG genes to mediate transcriptional silencing. Since binding of Pc to polytene chromosomes is abolished in H2Av mutant animals (Swaminathan, 2005), TIP60 complex-mediated histone variant exchange might cause the genetic interaction with PRC1. However, this study found that binding of PcG proteins to polytene chromosomes is unaffected in domino mutant larvae. It is possible that PRC1-mediated H2A ubiquitylation helps to recruit the TIP60 complex, whose histone acetylation or histone exchange activity assists in transcriptional repression. Alternatively, histone acetylation or exchange facilitates binding of the PRC1 complex to PREs. A similar mechanism has been invoked for the cooperation of the Esc-E(z) complex and PRC1, where Esc-E(z) trimethylates histone H3 lysine 27, which is recognized by the chromodomain of Polycomb (Qi, 2006).

This study has shown that the Drosophila TIP60 complex plays a role in epigenetic gene silencing in vivo. A similar case has been described for the yeast HAT complex SAGA (Spt-Ada-Gen5-acetyltransferase) that is required for both activation and repression of the ARG1 gene. Two other yeast HATs, Sas2 and Sas3, also promote gene silencing. Interestingly, the Drosophila HAT Chameau suppresses PEV and cooperates with PcG genes as well. TIP60, Sas2, Sas3, and Chameau are HATs that belong to the MYST family. Therefore, MYST family HATs in both yeast and flies can control epigenetic inheritance of silent chromatin (Qi, 2006).

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. The Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. These data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex (Kusch, 2004).

DNA double-strand breaks (DSBs) are a deleterious type of DNA damage leading to chromosomal breakage. Cells have developed mechanisms to detect and repair DSBs, which must access nucleosomal DNA. Two classes of activities regulate the accessibility of DNA by either covalently modifying histones or using adenosine triphosphate (ATP) hydrolysis to catalyze histone mobilization. Current knowledge suggests that covalently modified histones can create specific interaction sites for regulatory proteins and complexes (Kusch, 2004).

Incorporation of histone variants into nucleosomes provides another mechanism for altering chromatin structure. Whereas the major histones are assembled into nucleosomes during DNA replication, histone variants can be incorporated into chromatin in a replication-independent manner. An example of such an activity is the yeast Swr1p ATPase complex, which catalyzes the exchange of H2A for the variant H2A.Z in nucleosomes (Kusch, 2004).

Histone modifications can mark distinct chromatin locations. H2A.X, an essential mammalian histone variant required for genomic stability, becomes phosphorylated at sites of DSBs by conserved DNA damage-recognizing factors. Like H2A.X, H2A and H2Av become phosphorylated at DSBs in yeast and flies, respectively. Because repair requires access to DNA, it has been suggested that this phosphorylation might attract chromatin-remodeling complexes to DSBs. The removal of phospho-H2A.X is replication-independent and could be catalyzed by the same complexes. DSBs accumulate upon inactivation of the human Tip60 complex, implicating it as one candidate for a chromatin-remodeling complex with a role in DNA repair (Kusch, 2004).

This study demonstrates that the Drosophila dTip60 multiprotein complex catalyzes exchange of phospho-H2Av with unmodified H2Av. This reaction is catalyzed by two chromatin-dependent enzymes within the dTip60 complex: the histone acetyltransferase dTip60 and the ATPase Domino. These factors sequentially acetylate and then replace nucleosomal phospho-H2Av with H2Av from within the dTip60 complex (Kusch, 2004).

The dTip60 complex was purified from Drosophila S2 cells. dPontin, the fly homolog of a subunit of the human Tip60 complex, was epitope-tagged with a hemagglutin (HA)-Flag tag at the C terminus. The dPontinHAFlag-associated proteins were isolated from nuclear extracts by sequential Flag- and HA-affinity purification followed by a glycerol gradient. Peak fractions of dPontin-HAFlag, dTip60, and Domino were identified by immunoblotting and assayed for histone acetyltransferase activity. Several polypeptides that copurified with dPontinHAFlag were identified by multidimensional protein identification technology (MudPIT). This study identified polypeptides with homology to all 16 subunits of the human Tip60 complex. This analysis also revealed a substantial number of tryptic peptides from histones H2Av and H2B but not from other histones (Kusch, 2004).

Antibodies against dTip60, dMrg15, dTra1, dGas41, dIng3, and E(Pc) as well as against Domino, H2Av, and H2B were used in immunoblotting of gradient peak fractions and anti-dTip60 immunoprecipitates from nuclear extracts to confirm that these proteins are part of the dTip60 complex. dPontin-HAFlag stably associated with all dTip60 complex subunits examined, including dReptin, the fly homolog of the human Tip60 complex component Tip49b. Histones H2Av and H2B stably associated with the dTip60 complex, whereas histone H2A and other histones were not detected (Kusch, 2004).

Tip60 complexes function in DSB repair and contain the ATPase Domino/P400 and H2Av/H2B heterodimers. Because H2Av becomes phosphorylated at sites of DSBs, whether dTip60 complex remodeled nucleosomes containing phospho-H2Av was tested. Recombinant Drosophila nucleosomes were assembled containing H2Av with a point mutation that mimicked phosphorylation at Ser137 (Ser137 to Glu137; H2AvE). Upon incubation with the dTip60 complex, recombinant H2AvFlag/H2B heterodimers, acetyl-coenzyme A (acetyl-CoA), and ATP, a transfer of H2AvFlag to the nucleosomal arrays was observed. The transfer reaction proceeded rapidly (notable amounts of H2AvFlag were incorporated within 5 min) and depended on the presence of nucleosomes. Although relatively small amounts of H2AvFlag were transferred in the absence of ATP and/or acetyl-CoA, it was about seven times more efficient in the presence of both cofactors. Addition of a nonhydrolyzable ATP analog (gammaS-ATP) reduced the background activity of the complex. The dTip60 complex was highly selective for incorporation of H2Av into H2AvE-containing nucleosomal arrays. No H2AvEFlag was incorporated into nucleosomes containing H2Av, and no significant release of H2AvFlag was observed from nucleosomal arrays in the presence of H2AvEFlag/H2B heterodimers. Time course experiments revealed that the presence of acetyl-CoA enhanced the transfer speed and the quantity of H2Av incorporation. The incorporation rate of H2AvFlag into the nucleosomal arrays was unchanged when acetyl-CoA only was temporarily added to the exchange reactions and removed before the addition of heterodimers. This strongly suggests that the acetylation of the nucleosomal arrays by the dTip60 complex, but not of heterodimers, is crucial for optimal H2Av exchange (Kusch, 2004).

To examine the acetyltransferase specificity of the dTip60 complex, different combinations of recombinant histones as substrates in histone acetyltransferase (HAT) assays. In the presence of core histones, H2A, H2Av, and H2AvE were acetylated at equally low levels. However, in a nucleosomal context, acetylation of H2AvE was significantly increased over that observed for all other histones. This confirms that the dTip60 complex preferentially targets and acetylates phospho-H2Av in nucleosomes. In fact, Lys5 of histone H2Av is acetylated by the dTip60 complex. As individual monomeric histones, H2A, but not H2Av or H2AvE, was the preferred substrate of the dTip60 complex. By contrast, acetylation was about equal between H2A and H2Av when heterodimers with H2B were assayed, whereas acetylation of H2AvE was unchanged. Thus, dTip60 complex prefers H2Av-containing heterodimers over those containing H2AvE (Kusch, 2004).

Upon induction of DSBs, phospho-H2Av rapidly accumulates on chromatin with peak amounts after 10 to 15 min. During the course of DNA repair, this phosphorylation becomes undetectable within 180 min. The dTip60 complex acetylates and removes phospho-H2Av from nucleosomes in vitro. Thus, whether removal of phospho-H2Av during repair was dependent on dTip60 complex was tested in vivo. dTip60 or dMrg15 were depleted from S2 cells by RNA interference (RNAi). These cells were exposed to gamma irradiation to induce DSBs, and the nucleosomal histones were extracted after 0, 15, and 180 min. The amounts of H2Av and phospho-H2Av were compared by immunoblotting. In mock-treated cells, phospho-H2Av levels peaked after 15 min and were undetectable after 180 min. By contrast, phospho-H2Av levels remained high in cells depleted for either dTip60 or dMrg15. To confirm these findings in embryos, a null allele of dMrg15 was generated, and phospho-H2Av levels were tested after gamma irradiation. Again, the levels of phospho-H2Av remained higher in dMrg15 mutants than in wild-type embryos (Kusch, 2004).

Because the dTip60 complex acetylated nucleosomal phospho-H2Av in vitro, dependence of H2Av acetylation on dTip60 complex components was tested in vivo. Chromatin extracts were probed from gamma-irradiated double-stranded RNA (dsRNA)-treated S2 cells as well as dMrg15 mutant embryos with antibodies against H2A(acK5), which recognized H2Av(acK5). Transient acetylation of a protein band was detected that exhibits the migratory properties of phospho-H2Av. This acetylation was most prominent 15 min after gamma irradiation and was not detected in extracts of cells lacking dTip60 or dMrg15. Similar observations were made by immunolabeling dMrg15 mutant embryos. It is concluded that the dTip60 complex acetylates nucleosomal phospho-H2Av at Lys5 in a DSB-dependent manner (Kusch, 2004).

The Drosophila dTip60 complex is structurally homologous to its human counterpart. Both complexes share factors that are linked to cancer, transcription, and DNA repair, including Pontin, Reptin, Mrg15, Tra1, E(Pc), Gas41, and Tip60. The histone variant H2Av was detected within the Drosophila dTip60 complex. The human Tip60 complex is essential for DSB repair and regulation of apoptosis, two processes that have been linked to histone H2Av in flies. Also the yeast NuA4 complex appears to accumulate at DSBs (Kusch, 2004).

This study demonstrated that the Drosophila dTip60 complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. The histone-exchange reaction catalyzed by the ATPase Domino is enhanced by dTip60-mediated acetylation of nucleosomal phospho-H2Av. It appears likely that phospho-H2Av recruits the dTip60 complex to DSBs to facilitate chromatin remodeling during DNA repair. In yeast, the DNA damage-dependent H2A kinase Mec1 genetically interacts with subunits of the NuA4 complex, and cells missing NuA4 subunits are sensitive to DSB-inducing agents. The physiological roles of the dTip60-mediated phospho-H2Av removal at sites of DSBs could not be clearly separated from a potential function of this complex in DSB repair because of the intimate temporal link between DSB repair and phospho-H2Av clearance. However, the overexpression of phospho-H2Av did not induce G2/M arrest or affect DSB-dependent G2/M arrest, suggesting that this signal is not sufficient for damage checkpoint control (Kusch, 2004).

The loss of human Tip60 leads to the accumulation of DSBs and is linked to a growing number of cancer types. The histone variant H2A.X is essential for genomic stability and a candidate tumor suppressor. Thus, these findings help to understand the functional link between DNA damage-dependent H2A.X phosphorylation and the role of Tip60-type complexes during DSB repair in chromatin (Kusch, 2004).


REFERENCES

Search PubMed for articles about Drosophila Tip60

Baek, S. H., Ohgi, K. A., Rose, D. W., Koo, E. H., Glass, C. K. and Rosenfeld, M. G. (2002). Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein. Cell 110: 55-67. PubMed ID: 12150997

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Halkidou, K., Gnanapragasam, V. J., Mehta, P. B., Logan, I. R., Brady, M. E., Cook, S., Leung, H. Y., Neal, D. E. and Robson, C. N. (2003). Expression of Tip60, an androgen receptor coactivator, and its role in prostate cancer development. Oncogene 22: 2466-2477. PubMed ID: 12717424

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Kinoshita, A., Whelan, C. M., Berezovska, O. and Hyman, B. T. (2002). The gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein induces apoptosis via Tip60 in H4 cells. J Biol Chem 277: 28530-28536. PubMed ID: 12032152

Kusch, T., et al. (2004). Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306(5704): 2084-7. PubMed ID: 15528408

Lee, H. J., Chun, M. and Kandror, K. V. (2001). Tip60 and HDAC7 interact with the endothelin receptor a and may be involved in downstream signaling. J Biol Chem 276: 16597-16600. PubMed ID: 11262386

Leyssen, M., Ayaz, D., Hebert, S. S., Reeve, S., De Strooper, B. and Hassan, B. A. (2005). Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain. EMBO J 24: 2944-2955. PubMed ID: 16052209

Lin, Y. Y., Lu, J. Y., Zhang, J., Walter, W., Dang, W., Wan, J., Tao, S. C., Qian, J., Zhao, Y., Boeke, J. D., Berger, S. L. and Zhu, H. (2009). Protein acetylation microarray reveals that NuA4 controls key metabolic target regulating gluconeogenesis. Cell 136: 1073-1084. PubMed ID: 19303850

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Lorbeck, M., Pirooznia, K., Sarthi, J., Zhu, X., F. (2011). Microarray analysis uncovers a role for Tip60 in nervous system function and general metabolism. PLoS One 6: e18412. PubMed ID: 21494552

Ohno, K. and Sakurai, T. (2008). Orexin neuronal circuitry: role in the regulation of sleep and wakefulness. Front Neuroendocrinol 29: 70-87. PubMed ID: 17910982

Parisky, K. M., Agosto, J., Pulver, S. R., Shang, Y., Kuklin, E., Hodge, J. J., Kang, K., Liu, X., Garrity, P. A., Rosbash, M. and Griffith, L. C. (2008). PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit. Neuron 60: 672-682. PubMed ID: 19038223

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Pirooznia, S. K., Sarthi, J., Johnson, A. A., Toth, M. S., Chiu, K., Koduri, S. and Elefant, F. (2012a). Tip60 HAT activity mediates APP induced lethality and apoptotic cell death in the CNS of a Drosophila Alzheimer's disease model. PLoS One 7: e41776. PubMed ID: 22848598

Pirooznia, S. K., Chiu, K., Chan, M. T., Zimmerman, J. E. and Elefant, F. (2012b). Epigenetic regulation of axonal growth of Drosophila pacemaker cells by histone acetyltransferase tip60 controls sleep. Genetics 192: 1327-1345. PubMed ID: 22982579

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Biological Overview

date revised: 15 February 2015

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