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Transcriptional control of cognitive development
Transcriptional control of cognitive development Elizabeth J Hong, Anne E West and Michael E Greenberg Cognitive development is determined by both genetics and environment. One point of convergence of these two influences is the neural activity-dependent regulation of programs of gene expression that specify neuronal fate and function. Human genetic studies have linked several transcriptional regulators to neurodevelopmental disorders including mental retardation and autism spectrum disorders. Recent reports on two such factors, CREB-binding protein and methyl-CpG-binding protein 2, have begun to reveal how epigenetics and neuronal activity act to modulate the program of gene expression required for synaptic development and function. Addresses Division of Neuroscience, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA Corresponding author: Greenberg, Michael E ([email protected])
Current Opinion in Neurobiology 2005, 15:21–28 This review comes from a themed issue on Development Edited by Jane Dodd and Alex L Kolodkin Available online 26th January 2005 0959-4388/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2005.01.002
Introduction The postnatal period is a time of rapid changes in brain function. As infants explore their world, they acquire the cognitive and emotional capacities that constitute the essence of what it is to be human. Although the gross anatomy of the brain is largely established at birth, cognitive development during the postnatal period depends upon reorganizations in synaptic connectivity driven by sensory experience [1]. Several neurodevelopmental disorders of cognition manifest during this period, suggesting that they might primarily be diseases of synaptic maturation [2]. Interestingly, advances in human genetics have revealed that several of these disorders are caused by mutation of either sequence-specific DNA binding transcription factors or transcriptional cofactors (Table 1, [3]). Using animal models, researchers have begun to link molecular mechanisms of transcriptional regulation to cognitive dysfunction in two neurodevelopmental diseases, Rubinstein–Taybi (RTS) syndrome and Rett syndrome. Here, we review several recent studies that examine how www.sciencedirect.com
the gene products underlying these syndromes, the transcriptional regulators cAMP/Ca2+-responsive element binding (CREB)-binding protein (CBP, also known as CREBBP) and methyl-CpG-binding protein 2 (MeCP2), respectively, contribute to neuronal function. We also consider several emerging, but less well understood, cognitive disorders of transcriptional function. These studies highlight two important tenets of neural development: first, the key role epigenetic regulation of chromatin architecture plays in neuronal function, and second, the importance of neuronal activity in regulating the transcriptional programs that direct synaptic maturation.
Rubinstein–Taybi syndrome: CREB-binding protein RTS is a rare congenital disorder characterized by severe mental retardation, retarded growth, skeletal abnormalities, and an increased risk of cancer [4]. It is caused by heterozygous mutations of the CBP gene [5]. CBP and its closely related homolog p300 are multifunctional transcriptional coactivators that are recruited to gene promoters through association with a wide variety of sequencespecific DNA-binding transcription factors. In addition to acting as a molecular scaffold to facilitate the assembly of the basal transcription machinery, CBP possesses intrinsic histone acetyltransferase (HAT) activity that alters chromatin structure to make DNA more accessible to transcriptional machinery (Figure 1; [6,7]). In the brain, CBP is implicated in neuronal activity-dependent gene transcription, in part because it associates with the transcription factor CREB specifically when CREB becomes phosphorylated at Ser133. CREB Ser133 phosphorylation is induced by neuronal activity, is required for CREB activation, and is strongly correlated with forms of cognitive plasticity that require new gene expression. Furthermore, many gene targets of the activated CREB– CBP complex regulate synaptic function [8]. CBP, itself, is also a target of calcium-activated kinases in neurons [9,10]; however, the significance of CBP phosphorylation for its function in the brain remains unknown. Several mouse models have been generated to study the relationship between CBP and RTS. A conventional cbp heterozygous null exhibits many of the clinical features of RTS [11]. Mice carrying one allele of a truncated CBP protein that lacks the carboxy-terminal HAT domain exhibit a more severe RTS-like phenotype than that of the heterozygous null mice [12]. This observation is consistent with the hypothesis that this truncation mutant, which occurs in about 10% of RTS patients, has a dominant-negative effect on CBP function. Current Opinion in Neurobiology 2005, 15:21–28
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Table 1 Transcriptional regulators implicated in neurodevelopmental disorders. Gene
Function
Disease
ARX
Aristaless-related homeobox transcription factor
ATRX CACNA1C [55]
Chromatin remodeling helicase L-type voltage-gated calcium channel, drives activity-dependent transcription Transcriptional coactivator De novo genomic DNA methylation Kinase that potentiates steroid hormone-induced transcription Homolog of Drosophila transcription factor empty spiracles Transcription factor Transcriptional regulator in thyroid development Winged helix–loop–helix transcription factor Transcription downstream of hedgehog signaling pathway Transcriptional regulator Transcription factor Transcriptional corepressor Nuclear receptor binding transcriptional activator or coactivator Paired homeobox transcription factor Plant homeodomain-like transcription factor POU (Pit-1/Oct/Unc-86) domain transcription factor Transcriptional regulator A kinase that phosphorylates transcription factor CREB bHLH transcription factor, homolog of Drosophila single minded Homolog of Drosophila transcription factor sine oculus SRY-related transcription factor T-box transcription factor Transcriptional repressor Nuclear thyroid hormone receptor Transcriptional regulator Putative zinc finger transcription factor
Infantile spasms X-linked, Partington syndromes, X-linked lissencephaly with abnormal genitalia (XLAG) Syndromic and non-syndromic mental retardation Timothy syndrome (syndactaly, arrhythmia, and autism)
CBP DNMT3B DYRK1A [56] EMX2 FMR2 FOXE1 FOXP2 GLI3 GTF2I/GTF2RD1 HESX1 MECP2 NSD1 PAX8 PHF6 POU1F1 RAI1 RSK2 SIM2 SIX3 SOX3 TBX1 TGIF THRB TITF1 TRPS1 ZNF41 ZFHX1B ZIC2
Kruppel family zinc finger transcriptional repressor Zinc finger/homeodomain protein that binds the SMAD transcription factors Zinc finger transcriptional repressor
Rubinstein–Taybi syndrome Immunodeficiency, centromeric instability syndrome Down syndrome Schizencephaly X-linked nonspecific mental retardation Congenital hypothyroidism Developmental verbal dyspraxia Acrocallosal syndrome Williams syndrome Septooptic dysplasia Rett syndrome Sotos syndrome (cerebral gigantism) Thyroid dysgenesis Bo¨ rjeson–Forssman–Lehman syndrome Combined pituitary hormone deficiency Smith–Magenis syndrome Coffin–Lowry syndrome Down syndrome Holoprosencephaly 2 Mental retardation with growth hormone deficiency Velocardiofacial syndrome, DiGeorge syndrome Holoprosencephaly 4 Thyroid hormone resistance Congenital hypothyroidism Langer–Giedion syndrome, trichorhinophalangeal syndrome X-linked nonsyndromic mental retardation Hirschprung disease with microcephaly and mental retardation Holoprosencephaly 5
The table lists human genes encoding DNA-binding transcription factors and other transcriptional regulatory proteins that have been genetically implicated in a variety of neurodevelopmental disorders, including failures of cortical development, mental retardation, and autism spectrum disorders. Unless otherwise indicated, references can be found in Inlow and Restifo [3] or in the Online Mendelian Inheritance in Man database at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM.
These mouse models all exhibit grossly normal neuroanatomy, and three recent publications have explored the role of CBP function in the brain by way of behavioral and electrophysiological analyses [13,14,15]. In a comprehensive behavioral study, Alarcon et al. [13] observed that cbp haploinsufficient mice generally exhibit normal short-term memory, but defective long-term memory, in a variety of different tasks (Table 2). These animals also show abnormal late-phase long-term potentiation (l-LTP) in the hippocampus, but retain normal earlyphase LTP, which lasts only minutes and is independent of new gene expression. Similar defects in learning and memory tasks were seen in CBP truncation heterozygotes (Table 2; [12,14]). Gene rescue experiments suggest that the learning and memory phenotypes in CBP mutant mice are due, at least in part, to impaired CREB-dependent transcription. For example, the l-LTP deficit in cbp haploinsufficient mice Current Opinion in Neurobiology 2005, 15:21–28
can be partially rescued by overexpressing a constitutively active form of CREB [13]. Similarly, treatment with rolipram, a phosphodiesterase-4 inhibitor that enhances cAMP signaling and thus potentiates CREB activation, ameliorates l-LTP and long-term memory defects in cbp haploinsufficient mice and CBP truncation heterozygotes, respectively [13,14]. However, given that CREB target genes are expressed at normal basal levels and are appropriately induced by activity in cbp haploinsufficient mice [13], the phenotypic rescue might reflect a gain-of-function in the CREB pathway that compensates in parallel for the loss of other unidentified crucial CBP-mediated processes. One of the most difficult problems with conventional knockouts of ubiquitously expressed proteins, such as CBP, is the difficulty in distinguishing the participation of a protein in nervous system development from its acute requirement in neurons during synaptic function. To www.sciencedirect.com
Transcriptional control of cognitive development Hong, West and Greenberg 23
Figure 1
HDAC 1/2
HMT
Sin3A DNMT1 MeCP2
X
CH3 TGCGATC ACGCTAG CH3
Transcriptionally repressed
Transcriptionally permissive
Me Me Me Me Me Me Me
Me Me Me Me Me Me Me Me
HAT
Neuronal activity,
Ca2+ influx
Chromatin condensation
Ac
Ac
Ac
Ac
Ac
Ac Ac
Ac
Ac
Ac
CBP P P CREB
Pol II complex TBP
CRE
TATA Current Opinion in Neurobiology
A model of neuronal activity-mediated chromatin remodeling. In the basal state, the promoters of neural plasticity genes, such as Bdnf, could be methylated and bound by methyl-binding proteins, such as MeCP2, which are associated with a silencing complex that could include DNA methyltransferases (DNMT1), histone deacetylases (HDAC1/2), histone methyltransferases (HMT), and transcriptional co-repressors (Sin3A; [57]). These enzymatic activities modify the local chromatin to promote a repressive, transcriptionally non-permissive structure. Neuronal activity and calcium influx into the cell activate kinases for MeCP2, CREB, and CBP that induce the release of the MeCP2 transcriptional repression complex and the recruitment of CBP to phosphorylated CREB at the promoters of neural plasticity genes. CBP acts as a molecular scaffold to recruit the basal transcriptional machinery to sequence-specific transcriptional activators such as CREB. In addition, the histone acetyltransferase activity of CBP opens up local chromatin architecture making it permissive for transcription, thereby enabling enduring changes in gene expression that could underlie long-lasting forms of plasticity and long-term memory.
address the temporal and spatial requirement for CBP in learning and memory, the Mayford laboratory has recently generated an inducible cbp transgenic model. This mouse overexpresses a tetracycline-inducible allele of full-length CBP that bears two amino acid substitutions that abolish the HAT activity of CBP (CBP{HAT/}) [15]. Animals in which CBP{HAT/} transgene expression is activated only during adulthood exhibited longterm memory deficits, but no short-term memory deficits. These deficits can be fully rescued in a given animal by subsequent suppression of transgene expression, indicating that neurons are not irreversibly damaged by expression of mutant CBP{HAT/} [15]. In CBP{HAT/} transgenic animals, as well as cbp haploinsufficient mice, administration of histone deacetylase (HDAC) inhibitors transiently rescues the longterm memory deficit [13,15]. These data imply a role for the intrinsic HAT activity of CBP in memory consolidation. Alternatively, compensation might arise from the wide variety of basally repressed gene products that are ectopically induced by HDAC inhibitor treatment. Nevertheless, accumulating evidence suggests that epigenetic mechanisms, such as histone acetylation, www.sciencedirect.com
are important for neural plasticity and memory. Several groups have demonstrated that, in response to neuronal activity, changes in histone acetylation occur that correlate with the regulation of gene expression [16,17, 18,19,20]. However, the relative importance of histone acetylation for regulating brain function is poorly understood. Future studies face the challenge of abrogating HAT activity only at neural activity-regulated genes of interest to distinguish a specific role for histone acetylation in neuronal function and plasticity from the global role of histone acetylation in transcriptional control.
Rett syndrome: methyl-CpG-binding protein 2 Additional evidence that epigenetic mechanisms are crucial for proper cognitive development comes from studies of Rett syndrome, a relatively common X-linked neurodevelopmental disorder characterized by arrested neurological development and cognitive decline. Most cases of Rett syndrome are caused by mutations in the MECP2 gene [21,22], which belongs to a family of four methylCpG binding domain (MBD) proteins [23]. Essential for development, DNA methylation is regulated by developmental cues and environmental stimuli [24]. MBD Current Opinion in Neurobiology 2005, 15:21–28
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Table 2 Behavioral phenotypes of RTS mouse models. Mouse model
Developmental phenotype
Genotype for behavioral studies
Motor ability
Short-term memory (STM)
Long-term memory (LTM)
Rescue experiments
Conventional null
Homozygotes die at E10.5–E12.5 [58]; heterozygotes exhibit growth retardation and skeletal anomalies, but have a grossly normal neuroanatomy [11]
Heterozygote
Normal exploratory behavior, anxiety, motivation, prepulse inhibition [13]; defective motor learning in rotarod task [13]
Normal spatial working memory [13]; normal STM in object recognition task [13]; normal early LTP [13]
Defective LTM in both contextual and cued fear conditioning [13]; defective LTM in object recognition task [13]; no phenotype in Morris water maze, although mutant mice trend towards swimming slower [13]; defect in l-LTP [13]
Transgenic expression of a constitutively active form of CREB (VP16–CREB) or phosphodiesterase 4 inhibitors partially rescue l-LTP defect [13]; HDAC inhibitor administration enhances L-LTP in both wild type and mutant slices and reverses LTM defect in contextual fear conditiong task [13]
Insertional mutation into cbp allele leading to truncated CBP protein
Homozygotes die at E9.5–E10.5 [59]; heterozygotes exhibit more severe growth retardation and skeletal anomalies than haploinsufficient model, but grossly normal neuroanatomy [12]
Heterozygote
Hypolocomotion in dark [12]
Normal learning and working memory in Y-maze test [12] and object recognition task [12,14]
Defective LTM in step-through-type passive avoidance test and cued fear conditioning [12]; no phenotype in contextual fear conditioning or water maze [12]; defective LTM in object recognition task [14]
Phosphodiesterase 4 inhibitors restore LTM defect in object recognition task [14]
Regulated transgenic expressing a dominant-negative CBP transgene lacking HAT activity
Using the inducible tet (tetracycline) system under the control of the CaMKII promoter, transgene expression is limited to forebrain neurons only in adult life. No developmental phenotypes are reported, and mutants exhibit grossly normal neuroanatomy [15]
Heterozygote with activation of transgene only after 11 weeks of life
Normal anxiety and locomotor activity in open field activity test [15]; normal vision, motivation, and swimming ability in the visual platform version of Morris water maze [15]
Normal STM in object recognition task [15]
Defective LTM in object recognition task [15]; defective spatial LTM in the hidden platform version of Morris water maze [15]; no phenotype in contextual or cued fear conditioning tasks [15]
Transgene suppression rescues LTM defect in object recognition task [15]; HDAC inhibitor administration transiently rescues LTM defect in object recognition task [15]
proteins bind to methylated DNA within the genome and have been primarily studied for their role in long-term gene silencing [25]. MeCP2 is thought to repress transcription both by physically interfering with the recruitment of transcriptional activators and by recruiting histone-modifying enzymes that alter local chromatin structure, making it non-permissive for transcription. Three mouse models of Rett syndrome recapitulate key features of the disorder. Two of these are loss-of-function mutations of Mecp2 [26,27], whereas one bears a known disease-causing mis-sense mutation that results in expression of a truncated protein [28]. Interestingly, conditional deletion of Mecp2 in the brain recapitulates the phenotype of Mecp2 total null mutants, although neuronal dysfunction progresses more slowly in the conditional Mecp2 mutant [27]. Because MeCP2 is ubiquitously expressed, the apparently selective requirement for this protein in the brain remains mysterious. Given that MeCP2 is a core Current Opinion in Neurobiology 2005, 15:21–28
component of protein complexes that mediate gene silencing, the fact that deletion of Mecp2 leads to only subtle changes in gene expression is perplexing. Transcriptional profiling of human brain tissue from individuals with Rett syndrome identified some small perturbations in gene expression, but difficulties in collecting human samples and the effects of chronic illness complicate the interpretation of the results [29]. Microarray profiling of brain tissue from Mecp2 null mutant mice revealed no prominent changes in gene expression [30]. Several recent reports have identified the first targets of MeCP2 repression that could be relevant to the pathophysiology of Rett syndrome. Two studies [17,18] demonstrate that MeCP2 regulates the expression of the gene encoding brain-derived neurotrophic factor (BDNF), a secreted protein that has crucial roles in survival, development and synaptic plasticity in the nervous system [31]. Of the 8–10 alternative splice variants www.sciencedirect.com
Transcriptional control of cognitive development Hong, West and Greenberg 25
Figure 2
(a) Mouse Rat 5′
I
II
I
II
III
IV
V
III
IV 3′ Coding region
(b) Ca2+
Ca2+
Timothy CaMKII L-type voltage-sensitive calcium channels
Calmodulin Ras Raf MAPK Rsk2
NMDA-type glutamate receptors
Coffin–Lowry
CaMKIV Rett CBP: Rubenstein–Taybi
P
Ac H3
Ac
MeCP2
H3 CH3–CpG
Transcriptional coactivator(s) P?
CaRF CaRE1
P?
USF1/2 CaRE2
P P P
CREB CRE
Pol II Rat Bdnf exon III Mouse Bdnf exon IV
Current Opinion in Neurobiology
A schematic of signaling mechanisms mediating the activity-dependent activation of Bdnf. (a) Genomic organization of the Bdnf gene in mouse and rat. The Bdnf locus has a complex organization in which multiple promoters drive the expression of mRNAs containing alternative noncoding 50 exons spliced to a common downstream coding exon. Owing to the identification of an additional alternative 50 exon in the mouse Bdnf gene, exon III in rat is homologous to exon IV in mouse. For clarity, the rat exon nomenclature is used throughout this review. (b) A schematic showing the proximal promoter region to exon III (in rat) or exon IV (in mouse) that is signified by a star in (a). Rat Bdnf promoter III or mouse Bdnf promoter IV activation in response to calcium influx depends on the recruitment of at least three transcriptional activators, including calcium response factor (CaRF), upstream stimulatory factors (USFs), and CREB. These activators are believed to sit on the promoter, bound to specific DNA response elements (CaRE1/2 and CRE), poised to receive an activation signal [60]. Additionally, MeCP2 is bound to rat Bdnf promoter III or mouse Bdnf promoter IV in resting neurons, and dissociates in response to calcium influx to enable transcription of the exon [17,18]. Interestingly, CREB is maximally activated just minutes after membrane depolarization, whereas rat BDNF exon III transcripts are first detected at least 30 min later [32]. The delay could be due to a requirement for MeCP2 dissociation and/or CBP chromatin remodeling activity before exon III transcriptional initiation. Consistent with this idea, rat MeCP2 is phosphorylated with similarly delayed kinetics to rat Bdnf exon III induction [18]. Notably, the study of the signaling pathways mediating activity-dependent Bdnf induction has identified several key molecules that are mutated in genetic disorders of cognition, from channels at the membrane (e.g. Timothy syndrome) through kinases transducing the calcium signal (e.g. Coffin–Lowry syndrome) to the many transcriptional regulators highlighted in this review. Abbreviations: Ac, acetyl group; CaMKIV, calcium/calmodulin (CaM)-dependent protein kinase IV; CaMKII, calcium/calmodulin (CaM)-dependent protein kinase II; CaRE, calcium-response element [60]; CRE, cAMP/Ca2+-response element; H3, histone H3; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; Rsk2, p90 ribosomal S6 kinase 2.
expressed from the 4–5 alternative promoters at the Bdnf locus, those containing exons I and III in rat or exons I and IV in mouse are acutely transcribed in a calciumdependent manner in response to extracellular potassium-induced membrane depolarization [32,33]. Exon III in rat is homologous to exon IV in mouse (see Figure 2) and, for purposes of clarity, we will use the rat exon nomenclature in this review. Using chromatin immunoprecipitation, MeCP2 was found to be bound to the promoter region for exon III-containing Bdnf transcripts (Bdnf promoter III) in cultured rodent cortical neurons under basal conditions. Following membrane depolarization, which induces Bdnf exon III transcription, MeCP2 www.sciencedirect.com
dissociates from Bdnf promoter III, concurrent with histone modifications on Bdnf promoter III that promote transcription [17,18]. Consistent with a role for MeCP2 in Bdnf repression, Chen et al. [18] found that basal levels of Bdnf exon III transcripts in mutant Mecp2 mouse neurons are double those in wild type control cells. What is the mechanism of activity-dependent MeCP2 dissociation from Bdnf promoter III? Martinowich et al. [17] present evidence that membrane depolarization triggers CpG dinucleotide demethylation at Bdnf promoter III, whereas the data of Chen et al. [18] support a model in which membrane depolarization induces acute Current Opinion in Neurobiology 2005, 15:21–28
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phosphorylation of MeCP2, reducing its affinity for methyl CpG sequences within Bdnf promoter III. Chen et al. failed to detect a significant change in the methylation status of Bdnf promoter III, before or after membrane depolarization [18]. The discrepancy between these two reports might reflect the difference in the time course of membrane depolarization (90 min versus 72 h). Although no DNA demethylases have yet been definitively identified, the concept that rapid demethylation can occur and induce gene expression is supported by a recent study in T-lymphocytes that demonstrates active demethylation of the interleukin 2 promoter as early as 20 min following T-cell activation [34]. Future studies that detail the activity-dependent signaling pathways that modify MeCP2 functions should help to uncover the mechanisms by which neuronal activity and calcium influx trigger the release of MeCP2 from the Bdnf promoter. In addition to Bdnf, a recent study in the frog Xenopus laevis identified Hairy2a, a gene in the Delta–Notch signaling pathway, to be an MeCP2 target gene, and demonstrated that MeCP2 function is required for neurogenesis in Xenopus [35]. However, given that neuronal differentiation appears to be normal in Mecp2 mutant mice, and that expression of MeCP2 in the mammalian brain correlates with terminal differentiation, the relevance of this result to the mammalian nervous system remains to be established. Together, these studies provide new clues towards understanding the selective requirement for MeCP2 function in the brain. In contrast to the view that methyl-binding proteins primarily mediate long-term silencing of gene expression, MeCP2 appears to dynamically associate with at least a subset of its target promoters and to thereby regulate gene induction. When considered with the observation that the onset of neurological decline in individuals with Rett syndrome occurs during the first two years of life, these data suggest that MeCP2 might participate in the activity-dependent transcriptional program that directs postnatal synaptic maturation during the first years of life. The testing of this hypothesis will require dissecting the mechanism of MeCP2 activation in response to calcium influx, as well as the future identification of other activity-regulated MeCP2 target genes.
Other transcription factors associated with cognitive disorders Rett syndrome and RTS serve as examples of cognitive disorders that are relatively well understood at the molecular level. However, in most cases, how a given genetic lesion leads to neurological dysfunction is still not known. One such example is Williams syndrome (WS), which is characterized by an unusual cognitive profile of severe Current Opinion in Neurobiology 2005, 15:21–28
visual–spatial deficits with relative preservation of verbal ability [36,37]. WS is caused by a microdeletion on human chromosome 7 encompassing 24 genes, several of which function in the nervous system [38]. However, three recent genotype–phenotype correlations have strongly implicated hemizygosity of the transcription factors GTF2IRDI and GTF2I (also known as TFII-I) in the cognitive dysfunction of WS individuals [39–41]. Although the functions of GTF2IRDI and GTF2I in the brain are unknown, GTF2I is known to regulate the expression of c-Fos, an immediate early gene, the expression of which is strongly induced by neuronal activity [42]. The Bdnf gene also contains a putative GTF2I-binding element, although the functional role of this site has not been determined [33]. Recently, well-controlled structural and functional neuroimaging studies have identified distinct abnormalities in the dorsal visual–spatial processing stream that correlate with the WS cognitive deficit [43]. The identification of a neural substrate for WS dysfunction might now allow for the mapping of genetic factors underlying normal human visual–spatial cognition. Another intriguing link between transcription factors and cognitive function has been provided by the identification of mutations in the FOXP2 gene as the cause of an autosomal dominant disorder of speech and language abilities [44,45]. Foxp2 is a transcriptional repressor of the winged-helix/forkhead box (Fox) family [46]. In mouse and human, Foxp1 and Foxp2 are expressed in the developing and mature brain, at highest levels in the striatum and thalamus but also at a lower level in some cortical areas [47–49]. Recently, researchers pursuing the functional relevance of Foxp2 for vocalization have turned to songbirds, one of the few animal models available for studying auditory-guided vocal learning. In the zebra finch, Foxp2 expression in the striatal nucleus Area X, a critical nucleus of the vocal learning circuit, increases during the juvenile period when vocal learning occurs. Furthermore, in songbirds, such as the canary, which reestablish a new song annually, Foxp2 expression in Area X varies seasonally, with higher Foxp2 levels during months when the song pattern becomes unstable [50]. It is intriguing that Foxp2 expression is specifically detected in brain regions in the songbird that display plasticity, but Foxp2 expression is also detected in brain regions subserving motor function in mouse, bird, and human. Thus, whether FOXP2 dysfunction primarily affects circuits mediating higher language function [51,52] or disrupts motor planning [52–54] remains an open question.
Conclusions The examples discussed here highlight the importance of developing new animal paradigms for studying how transcription factor complexes regulate higher order cognitive functions. Further mechanistic understanding of neurodevelopmental disorders depends on identifying additional genes responsible for these disorders, identifying www.sciencedirect.com
Transcriptional control of cognitive development Hong, West and Greenberg 27
animal models in which behavior is affected by the relevant gene, and also acquiring better insight into the natural behaviors of genetically tractable animals such as the mouse. As the examples of CBP and MeCP2 illustrate, detailed investigation of animal models for neurodevelopmental disorders can yield important new insights into the molecular and cellular underpinnings of human cognition.
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10. Impey S, Fong AL, Wang Y, Cardinaux JR, Fass DM, Obrietan K, Wayman GA, Storm DR, Soderling TR, Goodman RH: Phosphorylation of CBP mediates transcriptional activation by neural activity and CaM kinase IV. Neuron 2002, 34:235-244. 11. Tanaka Y, Naruse I, Maekawa T, Masuya H, Shiroishi T, Ishii S: Abnormal skeletal patterning in embryos lacking a single Cbp allele: a partial similarity with Rubinstein–Taybi syndrome. Proc Natl Acad Sci USA 1997, 94:10215-10220. 12. Oike Y, Hata A, Mamiya T, Kaname T, Noda Y, Suzuki M, Yasue H, Nabeshima T, Araki K, Yamamura K: Truncated CBP protein leads to classical Rubinstein-Taybi syndrome phenotypes in mice: implications for a dominant-negative mechanism. Hum Mol Genet 1999, 8:387-396. 13. Alarcon JM, Malleret G, Touzani K, Vronskaya S, Ishii S, Kandel ER, Barco A: Chromatin acetylation, memory, and LTP are impaired in CBP+/S mice: a model for the cognitive deficit in Rubinstein–Taybi syndrome and its amelioration. Neuron 2004, 42:947-959. In a particularly comprehensive behavioral study, the authors provide the first electrophysiological and behavioral analysis of the haploinsufficient mouse model of RTS. In these cbp+/ mutant mice, some forms of longterm memory and l-LTP are impaired. Pharmacological inhibition of histone deacetylases potentiates l-LTP and restores the long-term memory deficit, suggesting a potential role for histone acetylation in neural plasticity and memory. 14. Bourtchouladze R, Lidge R, Catapano R, Stanley J, Gossweiler S, Romashko D, Scott R, Tully T: A mouse model of Rubinstein– www.sciencedirect.com
Taybi syndrome: defective long-term memory is ameliorated by inhibitors of phosphodiesterase 4. Proc Natl Acad Sci USA 2003, 100:10518-10522. 15. Korzus E, Rosenfeld MG, Mayford M: CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 2004, 42:961-972. The authors introduce a third mouse model for RTS, which inducibly expresses a mutant form of CBP lacking HAT activity (CBP{HAT/}) specifically in forebrain neurons. This genetic model is intended to dissociate any potential developmental contributions of CBP from its function in the mature nervous system, as well as to directly address whether or not CBP HAT activity, rather than its scaffolding function, is required for CBP function in the brain. CBP{HAT/} mutant mice are impaired in some forms of long-term memory, and the memory defect is reversible either by transgene suppression or by pharmacological inhibition of histone deacetylases, implicating the histone acetyltransferase activity of CBP in long-term memory function. 16. Huang Y, Doherty JJ, Dingledine R: Altered histone acetylation at glutamate receptor 2 and brain-derived neurotrophic factor genes is an early event triggered by status epilepticus. J Neurosci 2002, 22:8422-8428. 17. Martinowich K, Hattori D, Wu H, Fouse S, He F, Hu Y, Fan G, Sun YE: DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science 2003, 302:890-893. See annotation to Chen et al. [18]. 18. Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R, Greenberg ME: Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 2003, 302:885-889. This study and [17] identify the Bdnf gene as a mammalian target of MeCP2 repression. Using chromatin immunoprecipitation, the authors found that MeCP2 is bound to rat Bdnf promoter III (homologous to mouse Bdnf promoter IV) in resting cortical neurons, and, in response to membrane depolarization and calcium influx, MeCP2 dissociates from the promoter, concurrent with induction of the Bdnf transcript. Taken together, these studies pioneer the concept that a methyl-binding protein such as MeCP2 can dynamically associate with its target promoters and thereby regulate gene induction, in contrast to the prevailing dogma that methyl-binding proteins primarily mediate long-term gene silencing. 19. Guan Z, Giustetto M, Lomvardas S, Kim JH, Miniaci MC, Schwartz JH, Thanos D, Kandel ER: Integration of longterm-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure. Cell 2002, 111:483-493. This study performed using the mollusk Aplysia provides evidence that chromatin remodeling can take a role in the transcriptional program underlying long-term synaptic plasticity. 20. Tsankova NM, Kumar A, Nestler EJ: Histone modifications at gene promoter regions in rat hippocampus after acute and chronic electroconvulsive seizures. J Neurosci 2004, 24:5603-5610. 21. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY: Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 1999, 23:185-188. 22. Shahbazian MD, Zoghbi HY: Molecular genetics of Rett syndrome and clinical spectrum of MECP2 mutations. Curr Opin Neurol 2001, 14:171-176. 23. Wade PA: Methyl CpG-binding proteins and transcriptional repression. Bioessays 2001, 23:1131-1137. 24. Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003, 33:245-254. 25. Bird A: DNA methylation patterns and epigenetic memory. Genes Dev 2002, 16:6-21. 26. Guy J, Hendrich B, Holmes M, Martin JE, Bird A: A mouse Mecp2null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet 2001, 27:322-326. 27. Chen RZ, Akbarian S, Tudor M, Jaenisch R: Deficiency of methylCpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat Genet 2001, 27:327-331. Current Opinion in Neurobiology 2005, 15:21–28
28 Development
28. Shahbazian M, Young J, Yuva-Paylor L, Spencer C, Antalffy B, Noebels J, Armstrong D, Paylor R, Zoghbi H: Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron 2002, 35:243-254. 29. Colantuoni C, Jeon OH, Hyder K, Chenchik A, Khimani AH, Narayanan V, Hoffman EP, Kaufmann WE, Naidu S, Pevsner J: Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification. Neurobiol Dis 2001, 8:847-865. 30. Tudor M, Akbarian S, Chen RZ, Jaenisch R: Transcriptional profiling of a mouse model for Rett syndrome reveals subtle transcriptional changes in the brain. Proc Natl Acad Sci USA 2002, 99:15536-15541. 31. Poo MM: Neurotrophins as synaptic modulators. Nat Rev Neurosci 2001, 2:24-32. 32. Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME: Ca influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 1998, 20:709-726.
2+
33. Timmusk T, Palm K, Metsis M, Reintam T, Paalme V, Saarma M, Persson H: Multiple promoters direct tissue-specific expression of the rat BDNF gene. Neuron 1993, 10:475-489. 34. Bruniquel D, Schwartz RH: Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 2003, 4:235-240. This study convincingly demonstrates the active and rapid demethylation of the interleukin 2 promoter in T-lymphocytes in response to activation, both in vitro and in vivo. Methylation status was assessed by the bisulfite sequencing method. The change in methylation was found to be required for transcriptional activation, as assessed using transfected reporter plasmids. Although an enzymatic activity that is able to demethylate DNA has not yet been definitively identified in mammalian cells, this study provides evidence that the removal of DNA methylation can be a key event in gene activation. 35. Stancheva I, Collins AL, Van den Veyver IB, Zoghbi H, Meehan RR: A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos. Mol Cell 2003, 12:425-435. 36. Bellugi U, Lichtenberger L, Mills D, Galaburda A, Korenberg JR: Bridging cognition, the brain and molecular genetics: evidence from Williams syndrome. Trends Neurosci 1999, 22:197-207. 37. Tager-Flusberg H: Fulfilling the promise of the cognitive neurosciences. Neuron 2004, 43:595-596. 38. Tassabehji M: Williams–Beuren syndrome: a challenge for genotype–phenotype correlations. Hum Mol Genet 2003, 12:R229-R237. 39. Morris CA, Mervis CB, Hobart HH, Gregg RG, Bertrand J, Ensing GJ, Sommer A, Moore CA, Hopkin RJ, Spallone PA et al.: GTF2I hemizygosity implicated in mental retardation in Williams syndrome: genotype–phenotype analysis of five families with deletions in the Williams syndrome region. Am J Med Genet 2003, 123A:45-59. 40. Hirota H, Matsuoka R, Chen XN, Salandanan LS, Lincoln A, Rose FE, Sunahara M, Osawa M, Bellugi U, Korenberg JR: Williams syndrome deficits in visual spatial processing linked to GTF2IRD1 and GTF2I on chromosome 7q11.23. Genet Med 2003, 5:311-321.
44. Marcus GF, Fisher SE: FOXP2 in focus: what can genes tell us about speech and language? Trends Cogn Sci 2003, 7:257-262. 45. Lai CS, Fisher SE, Hurst JA, Vargha-Khadem F, Monaco AP: A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 2001, 413:519-523. 46. Shu W, Yang H, Zhang L, Lu MM, Morrisey EE: Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors. J Biol Chem 2001, 276:27488-27497. 47. Ferland RJ, Cherry TJ, Preware PO, Morrisey EE, Walsh CA: Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J Comp Neurol 2003, 460:266-279. 48. Takahashi K, Liu FC, Hirokawa K, Takahashi H: Expression of Foxp2, a gene involved in speech and language, in the developing and adult striatum. J Neurosci Res 2003, 73:61-72. 49. Teramitsu I, Kudo LC, London SE, Geschwind DH, White SA: Parallel FoxP1 and FoxP2 expression in songbird and human brain predicts functional interaction. J Neurosci 2004, 24:3152-3163. 50. Haesler S, Wada K, Nshdejan A, Morrisey EE, Lints T, Jarvis ED, Scharff C: FoxP2 expression in avian vocal learners and nonlearners. J Neurosci 2004, 24:3164-3175. Using the songbird as an animal model of vocal learning, this study identifies a correlation between Foxp2 expression and brain regions of the songbird that are involved in vocal learning. In the striatal nucleus Area X, a crucial nucleus in the vocal learning circuit, Foxp2 expression in zebra finch or canary increases specifically during times when the animal’s song pattern is unstable. This intriguing expression pattern suggests Foxp2 might be involved in the development or maturation of neuronal circuits underlying learned vocalizations. 51. Liegeois F, Baldeweg T, Connelly A, Gadian DG, Mishkin M, Vargha-Khadem F: Language fMRI abnormalities associated with FOXP2 gene mutation. Nat Neurosci 2003, 6:1230-1237. 52. Watkins KE, Vargha-Khadem F, Ashburner J, Passingham RE, Connelly A, Friston KJ, Frackowiak RS, Mishkin M, Gadian DG: MRI analysis of an inherited speech and language disorder: structural brain abnormalities. Brain 2002, 125:465-478. 53. Vargha-Khadem F, Watkins K, Alcock K, Fletcher P, Passingham R: Praxic and nonverbal cognitive deficits in a large family with a genetically transmitted speech and language disorder. Proc Natl Acad Sci USA 1995, 92:930-933. 54. Watkins KE, Dronkers NF, Vargha-Khadem F: Behavioural analysis of an inherited speech and language disorder: comparison with acquired aphasia. Brain 2002, 125:452-464. 55. Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, Napolitano C, Schwartz PJ, Joseph RM, Condouris K et al.: CaV1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 2004, 119:19-31. 56. Sitz JH, Tigges M, Baumgartel K, Khaspekov LG, Lutz B: Dyrk1A potentiates steroid hormone-induced transcription via the chromatin remodeling factor Arip4. Mol Cell Biol 2004, 24:5821-5834. 57. Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A: Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 1998, 393:386-389.
41. Gagliardi C, Bonaglia MC, Selicorni A, Borgatti R, Giorda R: Unusual cognitive and behavioural profile in a Williams syndrome patient with atypical 7q11.23 deletion. J Med Genet 2003, 40:526-530.
58. Tanaka Y, Naruse I, Hongo T, Xu M, Nakahata T, Maekawa T, Ishii S: Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of CREB-binding protein. Mech Dev 2000, 95:133-145.
42. Kim DW, Cochran BH: Extracellular signal-regulated kinase binds to TFII-I and regulates its activation of the c-fos promoter. Mol Cell Biol 2000, 20:1140-1148.
59. Oike Y, Takakura N, Hata A, Kaname T, Akizuki M, Yamaguchi Y, Yasue H, Araki K, Yamamura K, Suda T: Mice homozygous for a truncated form of CREB-binding protein exhibit defects in hematopoiesis and vasculo-angiogenesis. Blood 1999, 93:2771-2779.
43. Meyer-Lindenberg A, Kohn P, Mervis CB, Kippenhan JS, Olsen RK, Morris CA, Berman KF: Neural basis of genetically determined visuospatial construction deficit in Williams syndrome. Neuron 2004, 43:623-631.
Current Opinion in Neurobiology 2005, 15:21–28
60. West AE, Griffith EC, Greenberg ME: Regulation of transcription factors by neuronal activity. Nat Rev Neurosci 2002, 3:921-931.
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Current Opinion in Neurobiology 2005, 15:21–28 This review comes from a themed issue on Development Edited by Jane Dodd and Alex L Kolodkin Available online 26th January 2005 0959-4388/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2005.01.002
Introduction The postnatal period is a time of rapid changes in brain function. As infants explore their world, they acquire the cognitive and emotional capacities that constitute the essence of what it is to be human. Although the gross anatomy of the brain is largely established at birth, cognitive development during the postnatal period depends upon reorganizations in synaptic connectivity driven by sensory experience [1]. Several neurodevelopmental disorders of cognition manifest during this period, suggesting that they might primarily be diseases of synaptic maturation [2]. Interestingly, advances in human genetics have revealed that several of these disorders are caused by mutation of either sequence-specific DNA binding transcription factors or transcriptional cofactors (Table 1, [3]). Using animal models, researchers have begun to link molecular mechanisms of transcriptional regulation to cognitive dysfunction in two neurodevelopmental diseases, Rubinstein–Taybi (RTS) syndrome and Rett syndrome. Here, we review several recent studies that examine how www.sciencedirect.com
the gene products underlying these syndromes, the transcriptional regulators cAMP/Ca2+-responsive element binding (CREB)-binding protein (CBP, also known as CREBBP) and methyl-CpG-binding protein 2 (MeCP2), respectively, contribute to neuronal function. We also consider several emerging, but less well understood, cognitive disorders of transcriptional function. These studies highlight two important tenets of neural development: first, the key role epigenetic regulation of chromatin architecture plays in neuronal function, and second, the importance of neuronal activity in regulating the transcriptional programs that direct synaptic maturation.
Rubinstein–Taybi syndrome: CREB-binding protein RTS is a rare congenital disorder characterized by severe mental retardation, retarded growth, skeletal abnormalities, and an increased risk of cancer [4]. It is caused by heterozygous mutations of the CBP gene [5]. CBP and its closely related homolog p300 are multifunctional transcriptional coactivators that are recruited to gene promoters through association with a wide variety of sequencespecific DNA-binding transcription factors. In addition to acting as a molecular scaffold to facilitate the assembly of the basal transcription machinery, CBP possesses intrinsic histone acetyltransferase (HAT) activity that alters chromatin structure to make DNA more accessible to transcriptional machinery (Figure 1; [6,7]). In the brain, CBP is implicated in neuronal activity-dependent gene transcription, in part because it associates with the transcription factor CREB specifically when CREB becomes phosphorylated at Ser133. CREB Ser133 phosphorylation is induced by neuronal activity, is required for CREB activation, and is strongly correlated with forms of cognitive plasticity that require new gene expression. Furthermore, many gene targets of the activated CREB– CBP complex regulate synaptic function [8]. CBP, itself, is also a target of calcium-activated kinases in neurons [9,10]; however, the significance of CBP phosphorylation for its function in the brain remains unknown. Several mouse models have been generated to study the relationship between CBP and RTS. A conventional cbp heterozygous null exhibits many of the clinical features of RTS [11]. Mice carrying one allele of a truncated CBP protein that lacks the carboxy-terminal HAT domain exhibit a more severe RTS-like phenotype than that of the heterozygous null mice [12]. This observation is consistent with the hypothesis that this truncation mutant, which occurs in about 10% of RTS patients, has a dominant-negative effect on CBP function. Current Opinion in Neurobiology 2005, 15:21–28
22 Development
Table 1 Transcriptional regulators implicated in neurodevelopmental disorders. Gene
Function
Disease
ARX
Aristaless-related homeobox transcription factor
ATRX CACNA1C [55]
Chromatin remodeling helicase L-type voltage-gated calcium channel, drives activity-dependent transcription Transcriptional coactivator De novo genomic DNA methylation Kinase that potentiates steroid hormone-induced transcription Homolog of Drosophila transcription factor empty spiracles Transcription factor Transcriptional regulator in thyroid development Winged helix–loop–helix transcription factor Transcription downstream of hedgehog signaling pathway Transcriptional regulator Transcription factor Transcriptional corepressor Nuclear receptor binding transcriptional activator or coactivator Paired homeobox transcription factor Plant homeodomain-like transcription factor POU (Pit-1/Oct/Unc-86) domain transcription factor Transcriptional regulator A kinase that phosphorylates transcription factor CREB bHLH transcription factor, homolog of Drosophila single minded Homolog of Drosophila transcription factor sine oculus SRY-related transcription factor T-box transcription factor Transcriptional repressor Nuclear thyroid hormone receptor Transcriptional regulator Putative zinc finger transcription factor
Infantile spasms X-linked, Partington syndromes, X-linked lissencephaly with abnormal genitalia (XLAG) Syndromic and non-syndromic mental retardation Timothy syndrome (syndactaly, arrhythmia, and autism)
CBP DNMT3B DYRK1A [56] EMX2 FMR2 FOXE1 FOXP2 GLI3 GTF2I/GTF2RD1 HESX1 MECP2 NSD1 PAX8 PHF6 POU1F1 RAI1 RSK2 SIM2 SIX3 SOX3 TBX1 TGIF THRB TITF1 TRPS1 ZNF41 ZFHX1B ZIC2
Kruppel family zinc finger transcriptional repressor Zinc finger/homeodomain protein that binds the SMAD transcription factors Zinc finger transcriptional repressor
Rubinstein–Taybi syndrome Immunodeficiency, centromeric instability syndrome Down syndrome Schizencephaly X-linked nonspecific mental retardation Congenital hypothyroidism Developmental verbal dyspraxia Acrocallosal syndrome Williams syndrome Septooptic dysplasia Rett syndrome Sotos syndrome (cerebral gigantism) Thyroid dysgenesis Bo¨ rjeson–Forssman–Lehman syndrome Combined pituitary hormone deficiency Smith–Magenis syndrome Coffin–Lowry syndrome Down syndrome Holoprosencephaly 2 Mental retardation with growth hormone deficiency Velocardiofacial syndrome, DiGeorge syndrome Holoprosencephaly 4 Thyroid hormone resistance Congenital hypothyroidism Langer–Giedion syndrome, trichorhinophalangeal syndrome X-linked nonsyndromic mental retardation Hirschprung disease with microcephaly and mental retardation Holoprosencephaly 5
The table lists human genes encoding DNA-binding transcription factors and other transcriptional regulatory proteins that have been genetically implicated in a variety of neurodevelopmental disorders, including failures of cortical development, mental retardation, and autism spectrum disorders. Unless otherwise indicated, references can be found in Inlow and Restifo [3] or in the Online Mendelian Inheritance in Man database at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM.
These mouse models all exhibit grossly normal neuroanatomy, and three recent publications have explored the role of CBP function in the brain by way of behavioral and electrophysiological analyses [13,14,15]. In a comprehensive behavioral study, Alarcon et al. [13] observed that cbp haploinsufficient mice generally exhibit normal short-term memory, but defective long-term memory, in a variety of different tasks (Table 2). These animals also show abnormal late-phase long-term potentiation (l-LTP) in the hippocampus, but retain normal earlyphase LTP, which lasts only minutes and is independent of new gene expression. Similar defects in learning and memory tasks were seen in CBP truncation heterozygotes (Table 2; [12,14]). Gene rescue experiments suggest that the learning and memory phenotypes in CBP mutant mice are due, at least in part, to impaired CREB-dependent transcription. For example, the l-LTP deficit in cbp haploinsufficient mice Current Opinion in Neurobiology 2005, 15:21–28
can be partially rescued by overexpressing a constitutively active form of CREB [13]. Similarly, treatment with rolipram, a phosphodiesterase-4 inhibitor that enhances cAMP signaling and thus potentiates CREB activation, ameliorates l-LTP and long-term memory defects in cbp haploinsufficient mice and CBP truncation heterozygotes, respectively [13,14]. However, given that CREB target genes are expressed at normal basal levels and are appropriately induced by activity in cbp haploinsufficient mice [13], the phenotypic rescue might reflect a gain-of-function in the CREB pathway that compensates in parallel for the loss of other unidentified crucial CBP-mediated processes. One of the most difficult problems with conventional knockouts of ubiquitously expressed proteins, such as CBP, is the difficulty in distinguishing the participation of a protein in nervous system development from its acute requirement in neurons during synaptic function. To www.sciencedirect.com
Transcriptional control of cognitive development Hong, West and Greenberg 23
Figure 1
HDAC 1/2
HMT
Sin3A DNMT1 MeCP2
X
CH3 TGCGATC ACGCTAG CH3
Transcriptionally repressed
Transcriptionally permissive
Me Me Me Me Me Me Me
Me Me Me Me Me Me Me Me
HAT
Neuronal activity,
Ca2+ influx
Chromatin condensation
Ac
Ac
Ac
Ac
Ac
Ac Ac
Ac
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CBP P P CREB
Pol II complex TBP
CRE
TATA Current Opinion in Neurobiology
A model of neuronal activity-mediated chromatin remodeling. In the basal state, the promoters of neural plasticity genes, such as Bdnf, could be methylated and bound by methyl-binding proteins, such as MeCP2, which are associated with a silencing complex that could include DNA methyltransferases (DNMT1), histone deacetylases (HDAC1/2), histone methyltransferases (HMT), and transcriptional co-repressors (Sin3A; [57]). These enzymatic activities modify the local chromatin to promote a repressive, transcriptionally non-permissive structure. Neuronal activity and calcium influx into the cell activate kinases for MeCP2, CREB, and CBP that induce the release of the MeCP2 transcriptional repression complex and the recruitment of CBP to phosphorylated CREB at the promoters of neural plasticity genes. CBP acts as a molecular scaffold to recruit the basal transcriptional machinery to sequence-specific transcriptional activators such as CREB. In addition, the histone acetyltransferase activity of CBP opens up local chromatin architecture making it permissive for transcription, thereby enabling enduring changes in gene expression that could underlie long-lasting forms of plasticity and long-term memory.
address the temporal and spatial requirement for CBP in learning and memory, the Mayford laboratory has recently generated an inducible cbp transgenic model. This mouse overexpresses a tetracycline-inducible allele of full-length CBP that bears two amino acid substitutions that abolish the HAT activity of CBP (CBP{HAT/}) [15]. Animals in which CBP{HAT/} transgene expression is activated only during adulthood exhibited longterm memory deficits, but no short-term memory deficits. These deficits can be fully rescued in a given animal by subsequent suppression of transgene expression, indicating that neurons are not irreversibly damaged by expression of mutant CBP{HAT/} [15]. In CBP{HAT/} transgenic animals, as well as cbp haploinsufficient mice, administration of histone deacetylase (HDAC) inhibitors transiently rescues the longterm memory deficit [13,15]. These data imply a role for the intrinsic HAT activity of CBP in memory consolidation. Alternatively, compensation might arise from the wide variety of basally repressed gene products that are ectopically induced by HDAC inhibitor treatment. Nevertheless, accumulating evidence suggests that epigenetic mechanisms, such as histone acetylation, www.sciencedirect.com
are important for neural plasticity and memory. Several groups have demonstrated that, in response to neuronal activity, changes in histone acetylation occur that correlate with the regulation of gene expression [16,17, 18,19,20]. However, the relative importance of histone acetylation for regulating brain function is poorly understood. Future studies face the challenge of abrogating HAT activity only at neural activity-regulated genes of interest to distinguish a specific role for histone acetylation in neuronal function and plasticity from the global role of histone acetylation in transcriptional control.
Rett syndrome: methyl-CpG-binding protein 2 Additional evidence that epigenetic mechanisms are crucial for proper cognitive development comes from studies of Rett syndrome, a relatively common X-linked neurodevelopmental disorder characterized by arrested neurological development and cognitive decline. Most cases of Rett syndrome are caused by mutations in the MECP2 gene [21,22], which belongs to a family of four methylCpG binding domain (MBD) proteins [23]. Essential for development, DNA methylation is regulated by developmental cues and environmental stimuli [24]. MBD Current Opinion in Neurobiology 2005, 15:21–28
24 Development
Table 2 Behavioral phenotypes of RTS mouse models. Mouse model
Developmental phenotype
Genotype for behavioral studies
Motor ability
Short-term memory (STM)
Long-term memory (LTM)
Rescue experiments
Conventional null
Homozygotes die at E10.5–E12.5 [58]; heterozygotes exhibit growth retardation and skeletal anomalies, but have a grossly normal neuroanatomy [11]
Heterozygote
Normal exploratory behavior, anxiety, motivation, prepulse inhibition [13]; defective motor learning in rotarod task [13]
Normal spatial working memory [13]; normal STM in object recognition task [13]; normal early LTP [13]
Defective LTM in both contextual and cued fear conditioning [13]; defective LTM in object recognition task [13]; no phenotype in Morris water maze, although mutant mice trend towards swimming slower [13]; defect in l-LTP [13]
Transgenic expression of a constitutively active form of CREB (VP16–CREB) or phosphodiesterase 4 inhibitors partially rescue l-LTP defect [13]; HDAC inhibitor administration enhances L-LTP in both wild type and mutant slices and reverses LTM defect in contextual fear conditiong task [13]
Insertional mutation into cbp allele leading to truncated CBP protein
Homozygotes die at E9.5–E10.5 [59]; heterozygotes exhibit more severe growth retardation and skeletal anomalies than haploinsufficient model, but grossly normal neuroanatomy [12]
Heterozygote
Hypolocomotion in dark [12]
Normal learning and working memory in Y-maze test [12] and object recognition task [12,14]
Defective LTM in step-through-type passive avoidance test and cued fear conditioning [12]; no phenotype in contextual fear conditioning or water maze [12]; defective LTM in object recognition task [14]
Phosphodiesterase 4 inhibitors restore LTM defect in object recognition task [14]
Regulated transgenic expressing a dominant-negative CBP transgene lacking HAT activity
Using the inducible tet (tetracycline) system under the control of the CaMKII promoter, transgene expression is limited to forebrain neurons only in adult life. No developmental phenotypes are reported, and mutants exhibit grossly normal neuroanatomy [15]
Heterozygote with activation of transgene only after 11 weeks of life
Normal anxiety and locomotor activity in open field activity test [15]; normal vision, motivation, and swimming ability in the visual platform version of Morris water maze [15]
Normal STM in object recognition task [15]
Defective LTM in object recognition task [15]; defective spatial LTM in the hidden platform version of Morris water maze [15]; no phenotype in contextual or cued fear conditioning tasks [15]
Transgene suppression rescues LTM defect in object recognition task [15]; HDAC inhibitor administration transiently rescues LTM defect in object recognition task [15]
proteins bind to methylated DNA within the genome and have been primarily studied for their role in long-term gene silencing [25]. MeCP2 is thought to repress transcription both by physically interfering with the recruitment of transcriptional activators and by recruiting histone-modifying enzymes that alter local chromatin structure, making it non-permissive for transcription. Three mouse models of Rett syndrome recapitulate key features of the disorder. Two of these are loss-of-function mutations of Mecp2 [26,27], whereas one bears a known disease-causing mis-sense mutation that results in expression of a truncated protein [28]. Interestingly, conditional deletion of Mecp2 in the brain recapitulates the phenotype of Mecp2 total null mutants, although neuronal dysfunction progresses more slowly in the conditional Mecp2 mutant [27]. Because MeCP2 is ubiquitously expressed, the apparently selective requirement for this protein in the brain remains mysterious. Given that MeCP2 is a core Current Opinion in Neurobiology 2005, 15:21–28
component of protein complexes that mediate gene silencing, the fact that deletion of Mecp2 leads to only subtle changes in gene expression is perplexing. Transcriptional profiling of human brain tissue from individuals with Rett syndrome identified some small perturbations in gene expression, but difficulties in collecting human samples and the effects of chronic illness complicate the interpretation of the results [29]. Microarray profiling of brain tissue from Mecp2 null mutant mice revealed no prominent changes in gene expression [30]. Several recent reports have identified the first targets of MeCP2 repression that could be relevant to the pathophysiology of Rett syndrome. Two studies [17,18] demonstrate that MeCP2 regulates the expression of the gene encoding brain-derived neurotrophic factor (BDNF), a secreted protein that has crucial roles in survival, development and synaptic plasticity in the nervous system [31]. Of the 8–10 alternative splice variants www.sciencedirect.com
Transcriptional control of cognitive development Hong, West and Greenberg 25
Figure 2
(a) Mouse Rat 5′
I
II
I
II
III
IV
V
III
IV 3′ Coding region
(b) Ca2+
Ca2+
Timothy CaMKII L-type voltage-sensitive calcium channels
Calmodulin Ras Raf MAPK Rsk2
NMDA-type glutamate receptors
Coffin–Lowry
CaMKIV Rett CBP: Rubenstein–Taybi
P
Ac H3
Ac
MeCP2
H3 CH3–CpG
Transcriptional coactivator(s) P?
CaRF CaRE1
P?
USF1/2 CaRE2
P P P
CREB CRE
Pol II Rat Bdnf exon III Mouse Bdnf exon IV
Current Opinion in Neurobiology
A schematic of signaling mechanisms mediating the activity-dependent activation of Bdnf. (a) Genomic organization of the Bdnf gene in mouse and rat. The Bdnf locus has a complex organization in which multiple promoters drive the expression of mRNAs containing alternative noncoding 50 exons spliced to a common downstream coding exon. Owing to the identification of an additional alternative 50 exon in the mouse Bdnf gene, exon III in rat is homologous to exon IV in mouse. For clarity, the rat exon nomenclature is used throughout this review. (b) A schematic showing the proximal promoter region to exon III (in rat) or exon IV (in mouse) that is signified by a star in (a). Rat Bdnf promoter III or mouse Bdnf promoter IV activation in response to calcium influx depends on the recruitment of at least three transcriptional activators, including calcium response factor (CaRF), upstream stimulatory factors (USFs), and CREB. These activators are believed to sit on the promoter, bound to specific DNA response elements (CaRE1/2 and CRE), poised to receive an activation signal [60]. Additionally, MeCP2 is bound to rat Bdnf promoter III or mouse Bdnf promoter IV in resting neurons, and dissociates in response to calcium influx to enable transcription of the exon [17,18]. Interestingly, CREB is maximally activated just minutes after membrane depolarization, whereas rat BDNF exon III transcripts are first detected at least 30 min later [32]. The delay could be due to a requirement for MeCP2 dissociation and/or CBP chromatin remodeling activity before exon III transcriptional initiation. Consistent with this idea, rat MeCP2 is phosphorylated with similarly delayed kinetics to rat Bdnf exon III induction [18]. Notably, the study of the signaling pathways mediating activity-dependent Bdnf induction has identified several key molecules that are mutated in genetic disorders of cognition, from channels at the membrane (e.g. Timothy syndrome) through kinases transducing the calcium signal (e.g. Coffin–Lowry syndrome) to the many transcriptional regulators highlighted in this review. Abbreviations: Ac, acetyl group; CaMKIV, calcium/calmodulin (CaM)-dependent protein kinase IV; CaMKII, calcium/calmodulin (CaM)-dependent protein kinase II; CaRE, calcium-response element [60]; CRE, cAMP/Ca2+-response element; H3, histone H3; MAPK, mitogen-activated protein kinase; NMDA, N-methyl-D-aspartate; Rsk2, p90 ribosomal S6 kinase 2.
expressed from the 4–5 alternative promoters at the Bdnf locus, those containing exons I and III in rat or exons I and IV in mouse are acutely transcribed in a calciumdependent manner in response to extracellular potassium-induced membrane depolarization [32,33]. Exon III in rat is homologous to exon IV in mouse (see Figure 2) and, for purposes of clarity, we will use the rat exon nomenclature in this review. Using chromatin immunoprecipitation, MeCP2 was found to be bound to the promoter region for exon III-containing Bdnf transcripts (Bdnf promoter III) in cultured rodent cortical neurons under basal conditions. Following membrane depolarization, which induces Bdnf exon III transcription, MeCP2 www.sciencedirect.com
dissociates from Bdnf promoter III, concurrent with histone modifications on Bdnf promoter III that promote transcription [17,18]. Consistent with a role for MeCP2 in Bdnf repression, Chen et al. [18] found that basal levels of Bdnf exon III transcripts in mutant Mecp2 mouse neurons are double those in wild type control cells. What is the mechanism of activity-dependent MeCP2 dissociation from Bdnf promoter III? Martinowich et al. [17] present evidence that membrane depolarization triggers CpG dinucleotide demethylation at Bdnf promoter III, whereas the data of Chen et al. [18] support a model in which membrane depolarization induces acute Current Opinion in Neurobiology 2005, 15:21–28
26 Development
phosphorylation of MeCP2, reducing its affinity for methyl CpG sequences within Bdnf promoter III. Chen et al. failed to detect a significant change in the methylation status of Bdnf promoter III, before or after membrane depolarization [18]. The discrepancy between these two reports might reflect the difference in the time course of membrane depolarization (90 min versus 72 h). Although no DNA demethylases have yet been definitively identified, the concept that rapid demethylation can occur and induce gene expression is supported by a recent study in T-lymphocytes that demonstrates active demethylation of the interleukin 2 promoter as early as 20 min following T-cell activation [34]. Future studies that detail the activity-dependent signaling pathways that modify MeCP2 functions should help to uncover the mechanisms by which neuronal activity and calcium influx trigger the release of MeCP2 from the Bdnf promoter. In addition to Bdnf, a recent study in the frog Xenopus laevis identified Hairy2a, a gene in the Delta–Notch signaling pathway, to be an MeCP2 target gene, and demonstrated that MeCP2 function is required for neurogenesis in Xenopus [35]. However, given that neuronal differentiation appears to be normal in Mecp2 mutant mice, and that expression of MeCP2 in the mammalian brain correlates with terminal differentiation, the relevance of this result to the mammalian nervous system remains to be established. Together, these studies provide new clues towards understanding the selective requirement for MeCP2 function in the brain. In contrast to the view that methyl-binding proteins primarily mediate long-term silencing of gene expression, MeCP2 appears to dynamically associate with at least a subset of its target promoters and to thereby regulate gene induction. When considered with the observation that the onset of neurological decline in individuals with Rett syndrome occurs during the first two years of life, these data suggest that MeCP2 might participate in the activity-dependent transcriptional program that directs postnatal synaptic maturation during the first years of life. The testing of this hypothesis will require dissecting the mechanism of MeCP2 activation in response to calcium influx, as well as the future identification of other activity-regulated MeCP2 target genes.
Other transcription factors associated with cognitive disorders Rett syndrome and RTS serve as examples of cognitive disorders that are relatively well understood at the molecular level. However, in most cases, how a given genetic lesion leads to neurological dysfunction is still not known. One such example is Williams syndrome (WS), which is characterized by an unusual cognitive profile of severe Current Opinion in Neurobiology 2005, 15:21–28
visual–spatial deficits with relative preservation of verbal ability [36,37]. WS is caused by a microdeletion on human chromosome 7 encompassing 24 genes, several of which function in the nervous system [38]. However, three recent genotype–phenotype correlations have strongly implicated hemizygosity of the transcription factors GTF2IRDI and GTF2I (also known as TFII-I) in the cognitive dysfunction of WS individuals [39–41]. Although the functions of GTF2IRDI and GTF2I in the brain are unknown, GTF2I is known to regulate the expression of c-Fos, an immediate early gene, the expression of which is strongly induced by neuronal activity [42]. The Bdnf gene also contains a putative GTF2I-binding element, although the functional role of this site has not been determined [33]. Recently, well-controlled structural and functional neuroimaging studies have identified distinct abnormalities in the dorsal visual–spatial processing stream that correlate with the WS cognitive deficit [43]. The identification of a neural substrate for WS dysfunction might now allow for the mapping of genetic factors underlying normal human visual–spatial cognition. Another intriguing link between transcription factors and cognitive function has been provided by the identification of mutations in the FOXP2 gene as the cause of an autosomal dominant disorder of speech and language abilities [44,45]. Foxp2 is a transcriptional repressor of the winged-helix/forkhead box (Fox) family [46]. In mouse and human, Foxp1 and Foxp2 are expressed in the developing and mature brain, at highest levels in the striatum and thalamus but also at a lower level in some cortical areas [47–49]. Recently, researchers pursuing the functional relevance of Foxp2 for vocalization have turned to songbirds, one of the few animal models available for studying auditory-guided vocal learning. In the zebra finch, Foxp2 expression in the striatal nucleus Area X, a critical nucleus of the vocal learning circuit, increases during the juvenile period when vocal learning occurs. Furthermore, in songbirds, such as the canary, which reestablish a new song annually, Foxp2 expression in Area X varies seasonally, with higher Foxp2 levels during months when the song pattern becomes unstable [50]. It is intriguing that Foxp2 expression is specifically detected in brain regions in the songbird that display plasticity, but Foxp2 expression is also detected in brain regions subserving motor function in mouse, bird, and human. Thus, whether FOXP2 dysfunction primarily affects circuits mediating higher language function [51,52] or disrupts motor planning [52–54] remains an open question.
Conclusions The examples discussed here highlight the importance of developing new animal paradigms for studying how transcription factor complexes regulate higher order cognitive functions. Further mechanistic understanding of neurodevelopmental disorders depends on identifying additional genes responsible for these disorders, identifying www.sciencedirect.com
Transcriptional control of cognitive development Hong, West and Greenberg 27
animal models in which behavior is affected by the relevant gene, and also acquiring better insight into the natural behaviors of genetically tractable animals such as the mouse. As the examples of CBP and MeCP2 illustrate, detailed investigation of animal models for neurodevelopmental disorders can yield important new insights into the molecular and cellular underpinnings of human cognition.
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