SUMMARY
[En] The nervous system acts at the interface between an organism and its surrounding environment: the integration of incoming stimuli from the sensory systems results in long-lasting changes in neuronal circuitry that influence brain functions and behaviour. The modification of synaptic connections initiated by experience is referred to as plasticity and it requires a regulated programme of activity-dependent gene expression. A prominent role in transcriptional regulation is performed by epigenetic mechanisms, a vast set of enzymatic reactions altering chromatin structure.
To study the contribution of epigenetic factors in the regulation of plasticity, two complementary experimental systems are the early postnatal development of the visual cortex and the exposure of adult animals to environmental enrichment (EE).
In both these paradigms, experience alters cortical circuits at the anatomical and physiological level and elicits defined behavioural responses.
Light re-exposure after three days of dark rearing induces global histone H3 phosphorylation and histone H3-H4 acetylation in visual cortices of mice during the critical period, but not in adult animals. We recently found that methylation of lysine 4 in histone H3 is also increased by this protocol of visual stimulation. To characterize further these histone modifications we performed chromatin immunoprecipitation on the cAMP-responsive-element (CRE) at the promoter of the immediate-early gene c-fos and two activity-regulated microRNAs: miR132 and miR212. We found that histone H3 phosphoacetylation and methylation of lysine 4 is increased at the CRE of c-fos and miR132 after visual stimulation while miR212 CRE showed increased H3 acetylation only. This combination of histone marks correlates with the activation of transcription of these genes.
Histone acetylation has been demonstrated to mediate at least in part the effects of short-term exposure to EE. To gain further insight we performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIPSeq) to screen for genome-wide changes in acetylation of histone H3 elicited by EE in the mouse cortex. A computational analysis identified SINE repeats as a potentially relevant genomic element
that induces gene expression, possibly mediating the rearrangements of chromosomes inside the nucleus. The binding of transcription factors and histone modifications at SINEs has been validated for selected genes in primary cortical neurons. Finally, we performed reporter gene assay to provide a structure-to-function analysis of SINEs in activity-dependent gene expression in primary cortical neurons.
Alteration of the chromatin structure by epigenetic mechanisms is a fundamental process by which the nervous system is able to orchestrate the transcriptional programme necessary for plasticity of its synaptic connections throughout the whole life of an organism, thus allowing it flexibility in learning and behavioural responses.
[It] Il sistema nervoso consente ad un organismo animale di interagire con il proprio ambiente circostante mediante l’elaborazione delle informazioni provenienti dagli organi di senso e la modificazione a lungo termine dei propri circuiti neuronali, sia durante lo sviluppo che in età adulta. Tale plasticità delle connessioni sinaptiche in risposta all’esperienza si realizza attraverso un programma di espressione genica dipendente dall’attività neuronale. Un ruolo centrale nella regolazione dell’espressione genica è svolto da meccanismi epigenetici, un insieme di molteplici reazioni enzimatiche in grado di alterare la struttura della cromatina.
Lo sviluppo del sistema visivo durante il periodo critico e l’effetto dell’arricchimento ambientale su animali adulti rappresentano due sistemi sperimentali complementari con cui indagare i meccanismi epigenetici alla base della plasticità corticale. In entrambi questi paradigmi, infatti, l’esperienza altera i circuiti corticali a livello anatomico determinando precise risposte fisiologiche e comportamentali.
L’esposizione alla luce dopo tre giorni di allevamento al buio è in grado di indurre un aumento globale nei livelli di fosforilazione dell’istone H3 e di acetilazione degli istoni H3 ed H4, nelle cortecce visive di topi durante il periodo critico ma non in età adulta.
Recentemente abbiamo dimostrato che anche la metilazione dell’istone H3 a livello della lisina 4 viene indotta dal medesimo protocollo di stimolazione visiva. Per caratterizzare queste modificazioni istoniche in specifici promotori di geni transcritti in risposta all’attività elettrica abbiamo condotto esperimenti di immunoprecipitazione della
chromatina (ChIP) a livello di sequenze legate dal fattore di trascrizione CREB (cAMP-Responsive-Element Binding Protein). In risposta allo stimolo visivo si osserva un
aumento della fosfoacetilazione e metilazione dell’istone H3 sia nel promotore del gene c-fos che del microRNA, miR132 diversamente dal solo aumento di acetilazionde dell’istone H3 nel promotore di un altro microRNA attività-dipendente espresso nel sistema nervoso, il miR212. Queste combinazioni di modificazioni istoniche correlano con l’induzione della trascrizione di questi geni per effetto della riesposizione alla luce.
È stato dimostrato precedentemente che l’acetilazione degli istoni media, almeno in parte, gli effetti a breve termine dell’arricchimento ambientale.
Per poter analizzare la variazione dei livelli di acetilazione dell’istone H3 sull’intero genoma si è proceduto a sequenziare il DNA ottenuto mediante immunoprecipitazione della cromatina (ChIPSeq) da cortecce di topi sottoposti a tale stimolazione.
Un’analisi computazionale ha identificato elementi ripetitivi della famiglia delle SINE come potenziali mediatori dell’induzione di geni mediante un meccanismo di
rilocalizzazione dei loci genici all’interno del nucleo. Abbiamo validato il legame di fattori di trascrizione e le modificazioni istoniche a livello delle SINE in neuroni corticali primari. Inoltre, con saggi di espressione per un gene reporter abbiamo definito gli elementi di sequenza delle SINE necessari per indurre espressione di geni attività dipendente in neuroni corticali primari.
La regolazione della struttura della cromatina rappresenta un importante mediatore molecolare sfruttato dal sistema nervoso per orchestrare la trascrizione dei geni, in modo tale da adattare l’efficacia delle proprie connessioni sinaptiche all’influenza dell’esperienza durante l’intera vita di un organismo.
AIM OF THE PROJECT
I first developed an interest in the field of epigenetics in the central nervous system through a series of papers concerning the involvement of epigenetic mechanisms in the establishment of long-term neuropathological adaptations in the striatum of animals exposed to drugs of abuse {Hyman, Malenka & Nestler, 2006}. What appealed to me most about these studies was the idea of an evolutionary conserved system in the nucleus of neurons which can sense and respond to synaptic activity in order to orchestrate the transcription of genes and produce long-lasting changes in neuronal circuits. Regulated gene expression and nuclear dynamics per se represent an interesting subject of research for cellular biologists, and their analysis in neurons concerns understanding the language that genes and synapses use to communicate. This complex dialogue is thought to inform the way in which animals learn and store new memories, by remodelling their synapses. The field of epigenetics in the nervous system has branched out to draw on different aspects of neurobiology, and I have had the chance to work on the thrilling problem of how experience acts on gene expression via epigenetic mechanisms to modify cortical plasticity. I undertook two projects which explored this issue in two different physiological systems: the postnatal development of the visual cortex and the early effects of
environmental enrichment in the adult somatosensory cortex. In both of these systems, experience - whether visual stimulation or exploration of a novel enriched environment - can elicit anatomical, physiological and molecular changes which between them
contribute to the plasticity of the nervous system. A tightly regulated programme of gene expression is required for such processes to take place. Epigenetic mechanisms, and in particular post-translational modifications of histone tails, are integral to this regulatory network. I performed chromatin immunoprecipitation experiments to address how histone modifications and the binding of transcription factors to specific genomic
sequences are affected by experience in the visual and somatosensory cortices. Moreover, I analyzed how these events correlate with transcription of activity-dependent genes in vivo and in cultures of primary cortical neurons. The aim of my thesis is to define contribution of nuclear events to plasticity of the central nervous system.
INTRODUCTION
EPIGENETICS IN THE NERVOUS SYSTEM_A Form of Cellular Memory
From E. coli to elephants, living organisms are faced with the challenge of responding to an ever-changing environment on which they rely for survival. They have evolved different strategies to cope with this need, and animals in particular have developed behavioural responses and learning processes mediated by the nervous system.
Adaptation is a fundamental property of living organisms and every cell is able to respond to environmental signals by triggering molecular cascades, which generally lead to altered gene expression. Such a paradigm, initially described in pioneering studies on E.coli by J. Monod, F. Jacob and A. Lwoff, has informed the way we look at the regulation of gene expression up to the present day {Turner BM, 2009}. The logic behind the regulation of gene expression may be the same for bacteria and animals, but eukaryotic genomes
possess a different structure and organization. The highly complex and large size of higher eukaryotic genomes poses a puzzling question about how to achieve specific gene
regulation in response to environmental changes. At the interface between the action of the environment and the action of the genes, epigenetic mechanisms respond to signals and establish, reinforce, transmit and possibly even reverse programmes of gene expression {Bonasio et al, 2010}. No unambiguous definition of epigenetics exists but the concept behind it was clearly stated in the early days of the field by D.L. Nanney {1959}. Epigenetic regulative systems are envisaged as:
“ [..] signal interpreting devices, yielding predictable results in response to specific stimuli from inside and outside the cell. They are conceived as the integrative systems regulating the expression of genetic potentialities; mutual exclusion, simultaneity of expression, and adaptive cellular transformation could scarcely be achieved without efficient triggering devices. [..] An epigenetic change should not result in a permanent loss of information and a return to a previous condition of expression is always theoretically possible.” {Nanney D.L. Cold Spring Harbor Symp. Quant. Biol. 23: 327, 1959}
“ An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.” {Berger, 2009}
Epigenetic regulation is a widespread phenomenon across all living eukaryotic species and many of the pathways and molecules involved are conserved across the eukaryotic kingdom.
What is most relevant about this vast array of processes is that they confer a form of cellular memory by which regulatory networks responsible for cell specificity establish genomic signatures, which are inherited upon division or perpetuated over time. As has been stated in a recent review:
“ the epigenetic information resides in self-propagating molecular signatures that provide a memory of previously experienced stimuli, without irreversible changes in the genetic information” {Bonasio et al, 2010}.
Epigenetic mechanisms are employed by post-mitotic neurons as nuclear sensors of incoming stimuli and molecular signatures for long-lasting regulation of gene expression. They therefore participate in one of the most fundamental properties of the nervous system, namely the ability to alter the activity of its cells and the connections between them in response to the environment.
This property of neuronal circuits is called plasticity and it takes part in the development and functioning of the nervous system throughout the life of an organism. Plasticity of neuronal circuits allows for such diverse processes as learning, memory and adaptative behavioural responses.
Post-mitotic neurons utilize epigenetic mechanisms to integrate spatially and temporally disparate stimuli into self-sustaining transcriptional programmes which forms the basis of a form of cellular memory. This process is based on a bidirectional and dynamical
communication between a neuron’s synapses and its nucleus {Guan et al, 2002}.
Epigenetic mechanisms collectively refer to a number of enzymatic reactions that alter the chromatin, both the histones and DNA, and impart regulation over gene expression in response to appropriate signals, without irreversibly modifying the genetic content of a cell.
Epigenetic mechanisms work on different scales inside the nucleus to modify a large number of substrates such as N-tails of histones {Kouzarides, 2007}, cytidylate residues in the DNA and non-coding RNAs. Chromatin remodelling progresses from local, sequence-specific covalent marks on histone tails or CpG dinucleotides, through nucleosome
repositioning, DNA looping and loci relocalization, up to higher-order rearrangement of chromosomal territories inside the nucleus {Takizawa & Meshorer, 2008}.
Post-translational modifications of histone tails (PTMH), especially acetylation and
phosphorylation, and the methylation of CpG islands in the genome are examples of particularly relevant local chromatin modifications in neuronal cells. There is a wealth of other covalent modifications such as methylation of lysines and arginines, ubiquitination, sumoylation, ADP-ribosilation, palmitoylation, glycosylation and proline isomerization that are deposited along the tails of histones and for some of them there is evidence of involvement in neuronal functions {Franklin & Mansuy, 2010}. Such a richness of covalent marks has prompted researchers to decipher the so-called histone code, a sort of language in which PTMH are written, read and interpreted by nuclear proteins. The histone code contains the instructions to regulate the variety of processes taking place in the nucleus during the life of a cell. We can expect an even greater complexity hidden behind epigenetic regulation orchestrated by hundreds of enzymes catalyzing these reactions {Kouzarides T, Cell 2006} and multiple stimulus-dependent pathways leading to chromatin remodelling {in neurons: Riccio, 2010; Borrelli et al, 2008}.
IMPORTANCE OF EPIGENETIC REGULATION IN HEALTHY AND DISEASED BRAINS
Insights into the importance of epigenetic regulation in the brain came in part from the repertoire of pathological states affecting the nervous system which are caused by defects or dysfunctions in epigenetic regulators {Urdinguio RG et al, 2009}. These genetic
disorders are characterized by variable degrees of cognitive impairment, mental retardation and autism spectrum behaviours. Some examples of them are: Rubinstein-Taybi syndrome, in which acetyltransferase activity of coactivator CBP is defective {Petrij et al, 1995; Alarcón et al, 2004; Korzus et al, 2004}; X-linked mental retardation syndromes associated with a number of mutated loci including genes belonging to two different
families of histone demethylases, the PH2/PH8 and JARID1/JARID2 families
{Laumonnier et al, 2005; Siderius et al, 1999; Iwase et al, 2007} and neurodevelopmental Rett syndrome caused by mutations in methyl-cytosine binding protein 2 (MeCP2). The common feature shared by such variegated syndromes is the inability to maintain neuronal homeostasis due to the cumulative impact of inappropriate gene expression caused by altered chromatin regulation. A developing or learning brain could be
particularly sensitive to this because many routine cellular functions are performed while neurogenesis is occurring and synaptic connections are strengthened, remodelled and pruned {Ramocki & Zoghbi, 2008}.
The importance of epigenetic mechanisms in brain functions received support not only from studies on pathological conditions but also from a long list of experiments done on rodents. These studies demonstrated an epigenetic regulation of several physiological and behavioural processes such as learning and memory, the persistence of addiction states, the long-lasting and trans-generational effects of chronic stress and anxiety behaviours and the regulation of the circadian clock {Borrelli et al, 2008; Colvis et al, 2005; Jiang et al, 2008}.
Different learning paradigms have been useful to prove how epigenetic mechanisms are involved in the consolidation of long-lasting memories. Contextual fear conditioning, a hippocampus-dependent learning model, has been correlated with an increase in acetylation of histone H3 but not H4 (Levenson et al, 2004). This study was the first to indicate a link between the consolidation of long-term memories and epigenetic tagging of the genome. Dynamic regulation of histone acetylation by HDACs (histone deacetylases) and HATs (histone acetyltransferases) is required for synaptic plasticity and memory processes. Mice that are heterozygous for a dominant-negative form of truncated CBP
(CBPDN+/–) have significant deficits in various forms of long-term memory, including
step-through passive avoidance, novel object recognition and cued fear conditioning (Oike et al, 1999; Bourtchouladze et al, 2003). Two additional studies on inducible dominant or null allele of CBP demonstrated specific impairment in spatial learning, novel object
recognition and both contextual and cued fear memory {Korzus et al, 2004; Alarcon et al, 2004}. The effectiveness of HDAC inhibitors in restoring long-term memory suggests how
critical the balance between acetyltransferases and deacetylases is. Synaptic plasticity is able to elicit epigenetic changes similar to those that are involved in the formation of long-term memories. NMDA receptor-dependent glutamatergic transmission in the
hippocampus elicits acetylation and phosphorylation of H3 {Levenson et al, 2004} and the induction of late-phase LTP is significantly impaired in CBP+/- mice {Alarcon et al, 2004}. Administration of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) significantly improved the induction of late-phase LTP thus compensating for HAT haploinsufficiency in these mice. The prominence of epigenetic regulation in mediating long-term
adaptations in the brain is further confirmed by studies on reward-related learning, such as addiction to drugs of abuse in the circuitry linking nucleus accumbens, ventral
tegmental area and prefrontal cortex {Hyman, Malenga & Nestler, 2006}. In these studies, specific patterns of histone modifications such as histone H3 acetylation and methylation are detected in promoters of plasticity-related genes such as c-fos, Bdnf and CDK5 with different kinetics for primed (Bdnf, Cdk5) or desensitized genes (Fos) after acute and chronic cocaine administration {LaPlant, Nestler 2010; Kumar et al, 2005}.
LIFELONG CORTICAL PLASTICITY _ACTIVITY-DEPENDENT EPIGENETIC REGULATION DURING EARLY DEVELOPMENT AND IN ADULTHOOD
Plasticity of neuronal circuits is strongly dependent on patterns of activity, either
spontaneous or stimulus-driven. The first type of activity is especially prominent in the early stage of cortical development while inputs from the sensory systems continually shape cortical connections later on in life despite a progressive decline with age.
Experience-dependent plasticity takes over with the maturation of sense organs. Electrical activity driven by sensory inputs can be either permissive, to gate molecular programmes that establish new connections, or instructive, to shape and strengthen the efficacy of existing synapses {Sur & Rubenstein, 2005}. Physiological correlates of experience-dependent plasticity can be found in different sensory system but they have been most thoroughly studied in the mammalian visual system {Katz & Shatz, 1996}. Maturation of the synaptic connections in the primary visual cortex (V1) of mammals underlies
anatomical and physiological properties such as ocular dominance columns, orientation maps, visual acuity and orientation selectivity. Each of these properties may be set up “by
an early, intrinsic scaffold of connections” {Sur & Rubenstein, 2005} but it is subsequently shaped by visual activity. During early postnatal development there are time windows wherein manipulation of incoming stimuli are particularly effective in altering cortical circuitries because of a heightened plasticity of synaptic connections. Monocular deprivation (MD) and dark rearing (DR) are two well-established protocols for
manipulation of the visual stimulation. In the first case, animals are deprived of the visual inputs coming from one eye, for example by eyelid suture. In the second protocol, pups are reared in a dark environment which corresponds to a total deprivation of patterned visual stimulation. Dark rearing is known to prolong the critical period and to delay the maturation of visual acuity {Fagiolini et al, 1994} while altering synaptic structure {Wallace & Bear, 2004} and strength {Desain et al, 2002}. Periods of exposure to light during DR can trigger the process of visual development, therefore DR provides an experimental
platform in which to study the permissive role of activity on cortical plasticity and to define the underlying molecular mechanisms {Berardi et al, 2000}. An involvement of epigenetic mechanisms in the regulation of critical period plasticity has been previously suggested {Putignano et al, 2007}. The authors identified a candidate signalling and transcriptional mechanism which is linked to visually-induced histone modifications in the visual cortex only during the critical period. After the closure of the critical period, visual stimulation no longer drives changes in histone modifications. The functional relevance of this observation is suggested by restoration of ocular dominance plasticity in the adult mouse after treatment by histone deacetylases (HDAC) inhibitor, trichostatin A, performed during the period of monocular deprivation {Putignano et al, 2007}.
Plasticity of the nervous system is not exclusively confined to sensitive periods, but rather operates at all ages.
The ability to acquire new memories in humans at all ages is a trivial example of this statement, and experiments in adult rodents exposed to environmental enrichment have been particularly effective in providing details about the molecular correlates of enhanced plasticity in adulthood {Nithianantharajah & Hannan, 2006; van Praag et al, 2001}.
Environmental enrichment (EE) consists of housing animals in large groups, inside relatively spacious cages filled with a variety of objects (e.g. nesting materials, running
wheels, paper tunnels and wood blocks) that facilitate enhanced sensory, cognitive, motor and social stimulation relative to standard housing conditions {Sztainberg & Chen, 2010}. An essential component of this protocol of stimulation is the opportunity to reach high levels of voluntary physical activity on running wheels {Sale et al, 2008}. Enhanced exploration, cognitive activity, social interaction and physical exercise are all critical
aspects provided by environmental enrichment. Effects of EE at the anatomical level range from robust increases in: cortical thickness and weight, size of cell soma and nucleus, length of dendritic spines and synaptic size and number {Sale et al, 2008}.
Among the anatomical changes caused by EE there is neurogenesis in the dentate gyrus, synaptogenesis, gliogenesis and angiogenesis {ref 19, 46, 55 in van Praag G et al.}. EE also affects neurite branching and synapse formation in the cortex. Morphology of single synapses is modified in enriched animals too. These anatomical changes correlate with enhanced memory function in various learning tasks. Environmental enrichment has been associated with increased histone acetylation in the hippocampus and improved spatial memory. Additionally, administration of HDAC inhibitors enhanced associative learning in wild-type while in a mouse model of neuronal degeneration HDAC inhibition leads to recovery of inaccessible long-term memories {Fischer et al, 2007}. Thus, HDAC inhibitors act as enviromimetics because they mimic the effect of EE in improving spatial memory capacity in mice.
ACTIVITY-DEPENDENT SIGNALLING PATHWAYS TO CHROMATIN IN NEURONS
Plastic changes affecting synaptic connectivities are accompanied by induced transcription and synthesis of new proteins {Kandel ER, 2001; Flavell & Greenberg, 2008}. In the context of visual cortical plasticity, several large-scale analyses of gene expression have shown that DR affects expression of many target genes {Tropea et al, 2006; Majdan & Shatz, 2006} suggesting that a specific transcriptional programme is altered by visual deprivation. Similarly, in the adult cortex the effects of EE are accompanied by changes in gene
expression. Exposure to EE determines changes in expression levels of different classes of genes according to the duration of the stimulation. After a short period of exposure (3-6 hr) about half of the environmentally responsive, differentially expressed genes code for
proteins involved in macromolecule synthesis and processing, including transcription factors, translational regulatory enzymes, and enzymes involved in DNA, RNA, and protein processing. On the other hand, long term effects of EE on gene expression are associated with up-regulation of genes linked to neuronal structure, synaptic transmission and plasticity, neuronal excitability and neuroprotection {Rampon et al, 2000}.
Synaptic activity signals to the nucleus of neurons by triggering calcium entry through both somatodendritically and synaptically localized ion channels {Greer & Greenberg, 2008}. Different downstream transducers relay the signal to the nucleus via parallel or convergent signalling cascades. A major second messenger is cytosolic calcium whose levels rise in response to synaptic activity and which acts upstream of several different but interconnected signalling pathways (e.g. CaMKK cascade, MAPKK cascade, activation of RasGRF, PKA and many other transducers). Particularly important in the context of experience-dependent gene expression is the involvement of ERK (Extracellular-signal-Regulated Kinase) and the MAP kinase cascade. ERK is a key point of convergence for different signalling pathways: NMDAR-dependent CamKII activation, PKA activation and neurotrophin signalling {Berardi et al, 2003}. It has been shown that patterned vision is an effective activator of ERK in neurons of the visual cortex and its activation is required for ocular dominance shift induced by monocular deprivation during the critical period {Di Cristo et al., 2001}. Among the nuclear targets of ERK there are
c-AMP-responsive-element-binding protein (CREB) and histone H3. Transcription factor CREB has a pivotal role in various long-lasting forms of synaptic plasticity that are associated with learning and memory {West et al, 2002}. In the visual cortex of critical period mice, visual
stimulation after a period of dark rearing initiates an event of CREB-dependent gene expression {Cancedda et al, 2003}. Interestingly, the same protocol of stimulation is
ineffective in activating CREB later on in life when mice are no longer in the critical period {Putignano et al, 2007}. CREB is important for the coordinated expression of many activity-dependent genes in the brain. This fact explains its widespread role in neuronal growth, survival and synaptic plasticity. Among them Bdnf, c-fos and other genes involved in neurotransmission, cell structure, signal transduction, transcription, and metabolism. A distinctive feature of CREB-regulated genes is the presence of one or more consensus
elements (CRE) in their promoter regions which confer some degree of CREB-mediated regulation together with other cell-type and stimulus-specific activated transcription factors {Lonze&Ginty, 2002}. A prototypical CREB-regulated gene is the immediate-early gene c-fos, whose transcription in different types of neurons is rapidly but transiently induced by a wide variety of physiological stimuli (e.g. novel, visual, and social
experiences, circadian entrainment, fear conditioning, and exposure to drugs of abuse) {Greer & Greenberg, 2008}. Among the genes regulated by CREB there is a microRNA, miR132, which is rapidly induced upon stimulation and is responsible for dendritic outgrowth in hippocampal neurons {Wayman et al, 2008}. The authors show in the same paper that miR132 likely affects dendritic outgrowth through post-transcriptional
repression of p250GAP, a Rho family GTPase-activating protein in response to neuronal activity {Wayman et al, 2008}.
Activation of CREB by several kinases (e.g. RSK2/MSK, CaMKII and CaMKIV) which mediate phosphorylation of the critical residue serine-133 is a necessary step for the
recruitment of acetyltransferase CBP, in most of the cases {Lonze&Ginty, 2002}. CBP and its close homologue p300 are ubiquitously expressed transcriptional activator with a histone acetyltrasferase activity (HAT) and interact with several other neuronal transcription factors in addition to CREB: SRF, MEF2, c-jun and p53 {Goodman & Smolik, 2000}. The importance of CBP in the central nervous system (CNS) is underlined by its mutations in human patients of Rubistein/Taybi syndrome, a severe condition characterized by
cognitive dysfunctions. CBP itself can be phosphorylated by a number of extracellular signal-responsive Ser/Thr kinases such as CaMK-IV, RSK2 and ERK (p42/p44 mitogen-activated protein kinase). These kinases respond differently to calcium increase according to its route of entry into the neuron and differentially modulate the association of CBP with neuronal gene promoters {Hardingham et al, 1999}. HAT domain of CBP is necessary for its function in the CNS and it is involved in the consolidation of long term memories, as demonstrated by experiments with transgenic mice carrying an inducible dominant-negative CBP transgene that specifically blocks HAT activity {Korzus et al. 2004}. An interesting aspect of CBP distribution genome-wide emerges from a ChIPSeq screen done in depolarized primary cortical neurons which shows that activity-dependent CBP
binding marks neuronal enhancers. These regions are located more than 1Kb distal to known TSS and are situated within regions enriched in monomethylated lysine 4 in histone H3 {Kim et al, 2010}. RNA pol II is detected at basal levels at a subset of these extragenic enhancers but it becomes enriched upon stimulation suggesting a role for activity-dependent neuronal enhancers in recruitment of Pol II and organization of transcription.
In addition to acetylation, synaptic activity signals to chromatin by inducing other histone modifications, such as histone H3 phosphorylation {Riccio A, 2010}. This histone
modification is a good example of a direct link between signal transduction and
transcriptional regulation through chromatin remodelling. Mitogenic stimulation is known to induce rapid but transient phosphoacetylation of serine 10 of histone H3 which has been associated with transcriptional activation of immediate-early genes (IEGs) {Cheung et al, 2000; Nowak and Corces, 2000}. In a similar way to Ser133 phosphoacceptor residue in the transcription factor CREB, Ser10 in histone H3 represents a converging point of multiple intracellular pathways and kinases {Crosio et al, 2003}. In vivo experiments showed how dopaminergic, cholinergic and glutamatergic signalling pathways in hippocampal neurons induce phosphorylation of histone H3 in specific subfields of the hippocampus. There is also evidence for a synergistic effect of serine 10 phosphorylation on acetylation of the nearby lysine 14 by CBP and other HATs such as Gcn5, part of the SAGA complex {Merienne et al, 2001; Lo et al ,2000}. A functional correlation between acetylation and phosphorylation of these residues in histone H3 has also been
demonstrated in the visual cortex of critical period mice exposed to light after dark rearing {Putignano et al. 2007}. Activation of ERK leads to phosphorylation of MSK and
consequently to histone H3 phosphorylation. Such a pathway is activated by visual
stimulation only during the critical period, while in adult animals uncoupling mechanisms disconnect ERK and MSK activation, which is still responsive to light, from
CREB-depedent gene expression and histone modifications such as H3 acetylations and phosphorylation which are no longer induced.
GENOME-WIDE PROFILING OF HISTONE MODIFICATIONS SHED LIGHT ON TRANSCRIPTIONAL REGULATION OF GENES
Advancement in the technology to profile genome-wide patterns of histone modifications using massively parallel sequencing of immunoprecipitated DNA from ChIP experiments has facilitated understanding of the complex relationship between epigenetic marks and transcriptions.To analyze histone modification, all different technologies rely on a
procedure called chromatin immunoprecipitation (ChIP) in which chromatin is
immunoprecipitated with antibodies against a transcription factor, a chromatin-associated protein, or a modified histone {Bernstein et al, 2007}. PCR can be used to query for the presence of a predefined sequence in the immunoprecipitated genomic DNA but for more extensive genome-wide analyses different strategies have been designed. Tilling
oligonucleotide arrays can be employed to interrogate the entire non-repetitive portions of the human and mouse genome (ChIP-on-chip), while sequencing-based methods
(ChIPSeq) are an expanding technology which was initially limited by the large number of reads required. The approach consists of sequencing chromatin IP DNA and aligning the reads to the genome. The probability that a given genomic region was enriched in the chromatin IP is dependent on the number of sequencing reads that fall within the region. The number of reads is also a function of both the chromatin IP enrichment, strictly dependent on the antibody efficiency, and the desired resolution and accuracy of the detection {Bernstein et al, 2007}. Rapid technological developments in next-generation sequencing have made it possible to sequence tens or hundreds of millions of short DNA fragments in a single run {Park PJ, 2009}. ChIPSeq has become a promising technology for its higher resolution, fewer artifacts, greater coverage and a larger dynamic range than ChIP-chip.
The implementation of chromatin immunoprecipitation coupled to high-throughput sequencing allows comprehensive assessment of chromatin modifications across the genome to identify the fraction of the genome subject to experience-driven epigenetic plasticity in the adult CNS {Roth & Sweatt, 2009}. Few reports using this technique in the nervous system have been published so far. Recently, researchers identified activity-dependent neuronal enhancers by means of ChIPSeq targeting binding of CBP and other
transcription factors in conjunction with a panel of different histone modifications {Kim et al, 2010}. A second paper used ChIPSeq to demonstrate that the severe abolishment of learning-induced gene expression in the hippocampus of aging mice is linked - at least in part - to deregulated H4K12 acetylation along the coding regions of genes that are
normally upregulated upon fear-conditioning in young, healthy mice {Peleg et al, 2010}. Apart from these two published reports this genome-wide approach to study dynamical experience-dependent chromatin remodelling has not been exploited in the nervous system yet.
An important and thrilling question to answer by using this technique is how stimulus-driven signalling events regulate neuronal transcription through epigenetic mechanisms. This is not an easy task to undertake but some promising results have been obtained in other cell types from genome-wide maps of histone modifications, occupancy of Pol II machinery and cell-type specific transcription factors. A series of experiments performed on CD4+ T lymphocytes defined a high-resolution map of DNaseI hypersensitive sites which was used as scaffold on which to combine and analyze data for RNA pol II binding, patterns of histone modifications relatively to the TSS of genes and their corresponding expression levels {Boyle et al, 2008}. Genome-wide distribution of many histone
methylation {Barski et al, 2007} and acetylation marks {Wang et al 2008} in this resting lymphocytes confirmed the notion that chromatin modifications play a role in gene expresssion {Schones & Zhao, 2008}. Histone H3 lysine 4 methylation and histone
acetylation positively correlate with transcriptional levels, are highly enriched in promoter regions and some of them extend significantly into the transcribed regions {Liu et al, 2005; Pokholok et al, 2005}. Many activating modifications tend to cluster at key regulatory regions, which might reinforce active chromatin states and provide potential redundancy in the system. However, the picture is certainly more complex than this. In fact, silent genes sometimes bear marks of transcriptional activation such as H3K4me3. Studies in yeast suggest that some histone modifications result from active transcription {Ng et al, 2003} and have been proposed to be a memory of past transcriptional activation {Ng et al, 2003}. Experiments in mice have also suggested a different scenario in which chromatin modifications might precede changes in gene expression {Chambeyron and Bickmore,
2004}. Recent findings have further confirmed that inducible genes are poised for activation by chromatin in resting human T cells {Barski et al, 2009}.
DISCUSSION
HISTONE POST-TRANSLATIONAL MODIFICATIONS ARE MEDIATORS OF
EXPERIENCE-DEPENDENT GENE EXPRESSION IN THE VISUAL CORTEX DURING THE CRITICAL PERIOD
Results from this study illustrate a further case of how experience-dependent neuronal activity drives gene expression and histone modifications in vivo. Histone
post-translational modifications (HPTMs) are relevant to visual cortical plasticity as well as to other forms of brain plasticity, such as hippocampal-dependent memory formation {Roth TL & Sweatt JD, 2009} or striatum-dependent drug addiction {Laplant Q & Nestler EJ, 2010}. Prevalence of epigenetic mechanisms in brain functions indicates how nuclear processes regulating coordinated gene expression are necessary for physiological response of the CNS to environmental stimuli {Dulac C, 2010}.
Administration of histone deacetylase inhibitors (HDACi) is able to promote enhanced visual cortical plasticity in adult rodents tested for ocular dominance plasticity or for recovery of normal visual acuity of an amblyopic eye {Putignano et al., 2007; Silingardi et al., 2010}. The effect of these agents is to alter the global balance between HATs and HDACs, in favour of increased histone acetylation levels. Applied to other protocols of cortical plasticity inhibition of HDACs generally boosts plastic adaptations in brain circuits leading to improved outputs in learning and memory tasks and recovery from age-dependent memory impairment {Fisher et al. 2007; Peleg et al., 2010}.
It has been previously suggested that a developmental downregulation of epigenetic mechanisms, consisting in covalent modifications of histone tails, gate the potential for plasticity in the visual cortex in response to sensory experience {Putignano et al., 2007}. A common set of plasticity-related genes are readily activated by visual stimulation during the critical period while they are kept repressed, likely through epigenetic mechanisms, later in life {Tropea et al., 2006; Majdan & Shatz, 2006}. Even though we ignore the precise target genes modulated by HDACs we can envisage that their inhibitors act in the adult visual cortex to release a brake on gene expression activated by sensory stimulation.
Administration of HDACi could target enzymatic activities at specific chromosomal loci where members of this class of histone modifiers are targeted by association in
co-repressor complexes {Riccio A, 2010}. Despite their broad spectrum of action only a small proportion of genes display altered transcription in response to HDACi, and interestingly, among the genes that do change, downregulation is as common as upregulation {Peart et al. 2005}. It is becoming clearer that relevant alteration in histone acetylation levels could occur in intergenic regions rather than at gene promoters {Turner BM, 2009; Crepaldi et al. unpublished data}.
According to our results another histone tail modification which is downstream of visually regulated signalling pathways is methylation of histone H3. Histone methylation is even a richer and more complex histone mark compared to histone acetylation {Borrelli et al., 2008} but for which we know less in terms of enzymatic regulation and its role in long-term memory formation or the behavioural effects of its dysregulation. There is an increasing interest toward understanding how histone methylation affects neuronal function and plasticity and accumulating evidences are pointing to a prominent role for this histone mark {Akbarian & Huang, 2009}. Recently discovered histone demethylases {Shi & Whetstine, 2007} changed a well-established paradigm by which histone
methylation was a long-lasting, quite static mark. Presently, many different histone methyltransferases (HMT) and demethylases (HDMT) are known to exist {Kouzarides, 2007} and many of them are expressed in the nervous system {Akbarian & Huang, 2009}. Moreover, mutations in several of these enzymes, such as GLP/G9a, a H3K9-specific HMT and JARID1C/SMCX a H3K4-specific HDMT, have been linked to mental retardation and autism-spectrum disorders in humans {Raymond, 2006; Kramer & Bokhoven, 2008;
Kleefstra et al. 2006; Adegbola et al. 2008; Iwase et al, 2007}.
There is an intrinsic richness in this histone mark due to its depositions at many different sites (lysine and arginines), with different degrees of methylation (mono-, di- or
trymethylation), by opposing enzymatic activities (HMTs or HDMTs) which overall cooperates in generating a combinatorial histone code that is read by proteins containing specialized domains such as chromodomains of PHD fingers {Ruthenburg et al, 2007}.
Methylation of lysine 4 on histone H3 appears to be a particularly interesting target
because of specific HTMs and HDMTs acting at this site, such as MLL1 methyltransferase, LSD1 and JARID1C demethylases, and the crosstalk with another relevant methylation in the CNS, that of lysine 9 in histone H3 {Kleefstra et al. 2006; Schaefer et al, 2009; Maze et al. 2010}. Remarkably, this crosstalk can be mediated by a single enzyme, LSD1, which has a dual substrate specificity and it can demethylate either lysine 4 or lysine 9 depending on the composition of its recruiting complex, thus acting both as a repressor or a
co-activator {Wang et al. 2007; Shi & Whetstine, 2007}. A specific splice-variant of this enzyme is restricted to the CNS and its expression is fine-tuned within the perinatal period when it is likely required for early neurite morphogenesis {Zibetti et al. 2010}. It would be
interesting to further explore the role of histone methylation in visual cortical plasticity and in particular the involvement of different enzymatic complexes in fine-tuning of this histone mark during development. Moreover, it would be worth addressing how
pharmacological intervention altering this epigenetic mark is affecting adult cortical plasticity.
Expression of miR132 is strongly affected by visual experience during the critical period. We demonstrated this by manipulation of visual inputs such as dark rearing (DR) and monocular deprivation (MD). Both types of intervention downregulated the levels of the mature microRNA suggesting that visual experience is permissive in regulation of its expression. But what could be the mechanisms behind induction of miR132 upon visual stimulation?
We propose that visual experience regulates miR132 levels mainly at the transcriptional level through a combination of activating histone marks at a CRE regulatory site bound by CREB and which is necessary for proper promoter activity in response to neurotrophic stimulation {Remenyi et al. 2010}. Further evidence supports to existence of a visually-induced transcriptional switch. A related microRNA, miR212, which is associated in a genomic cluster with miR132 and has its own proximal CRE sequence, does not respond to light re-exposure after dark rearing as observed for miR132. Three days of DR
expression. This lack of induction correlates with the absence of the histone marks that we detected at the CRE of miR132. In addition to this, we quantified levels of the Pri-miR132 in the visual cortex of critical period mice after DR and MD to find the same pattern of regulated expression seen with the mature sequence. We cannot exclude the contribution of other mechanisms in experience-dependent regulation of miR132 levels in the visual cortex, such as regulated stabilization of this microRNA as described for light-adaptation in the retina {Krol et al. 2010}. Despite this fact our data strongly support transcriptional and epigenetic mechanisms in experience-dependent regulation of miR132 expression.
MiR132 REGULATES DIVERSE PROCESSES WHICH AFFECTS STRUCTURAL PLASTICITY IN THE CENTRAL NERVOUS SYSTEM
After an initial characterization of miR132 as one of most prominent CREB-regulated genes in cultured cortical neurons and its implication in morphogenesis and dendritic plasticity {Vo et al. 2005; Wayman et al. 2008} recently published papers validated its role in structural plasticity in vivo {Edbauer et al. 2010; Impey et al. 2010}.
miR132 is required for dependent dendritic growth and promotes
activity-dependent increase in spine density and protrusion size by downregulating p250GAP and activating the Rac1-PAK pathway {Impey et al., 2010}. MiR132-p250GAP circuit modulates synapse-specific Kalirin7-Rac1 signalling leading to actin remodeling and spine formation. Furthermore, overexpression of miR132 in hippocampal neurons increased the formation of stubby and mushroom spine with high protrusion width and enhanced synaptic strength {Edbauer et al., 2010}. Conversely, knock-down of miR132 expression caused dendritic arbour pruning {Edbauer et al., 2010}, reduced spine formation and excitatory synaptic transmission {Impey S et al., 2010} and reduced spine volume {Siegel et al. 2009}. These studies are relevant to visual cortical plasticity since we found that monocular deprivation during the critical period downregulates miR132 expression in the visual cortex controlateral to the deprived eye. The same type of visual manipulation also decreases dendritic spine stability {Mataga et al. 2004; Oray et al., 2004} mirroring the effect of miR132 loss-of-function experiments.
An additional target gene of miR132 is transcriptional co-regulator MeCP2 {Klein et al., 2007}, which binds methylated cytidylate residues in CpG islands and it is involved in pathogenesis of Rett syndrome. The longest 3’ UTR of miR132 contains conserved MREs for miR132. Expression of 2’-O-methyl blockers of miR132 is able to increase MeCP2 levels under basal condition. Activity-induced CREB-dependent miR132 expression indirectly downregulate Bdnf through intermediate repression of MeCP2 which in turn acts as a coactivator for Bdnf-exon III expression. miR132 is also known to be positively induced by neurotrophic stimulation {Vo et al. 2005}. Therefore, miR132 is involved in a regulatory loop which maintains homeostatic levels of MeCP2 through regulation of Bdnf expression. All three molecular players have been implicated in postnatal neuronal maturation and synaptogenesis and their levels are altered in mouse models of Rett syndrome {Ramocki & Zoghbi, 2008}. It has been shown that miR132 action on spine morphology involves its interaction with FMRP. FMRP is a RNA-binding protein, that associates with
polyribosomes, suppresses translation of specific mRNAs and whose mutation by unstable trinucleotides expansion causes fragile X syndrome. Role of miR132 in this trancriptional regulatory network could be even more complex in light of a role for associated miR212 in amplifying the activity of CREB in the dorsal striatum of mice with extended access to cocaine consumption {Hollander et al., 2010}. Mir212 action occurs through Raf1-mediated sensitization of adenylyl cyclase activity and increased TORC expression.
Considering the functional relevance of CREB-dependent gene expression in visual
cortical plasticity it would be tempting to investigate how visual experience acts upon this complex regulatory loop.
