Regulation of neuronal gene expression is critical to central nervous system development. Here, we show that REST regulates the transitions from pluripotent to neural stem/progenitor cell and from progenitor to mature neuron. In the transition to progenitor cell, REST is degraded to levels just sufficient to maintain neuronal gene chromatin in an inactive state that is nonetheless poised for expression. As progenitors differentiate into neurons, REST and its co-repressors dissociate from the RE1 site, triggering activation of neuronal genes. In some genes, the level of expression is adjusted further in neurons by CoREST/MeCP2 repressor complexes that remain bound to a site of methylated DNA distinct from the RE1 site. Expression profiling based on this mechanism indicates that REST defines a gene set subject to plasticity in mature neurons. Thus, a multistage repressor mechanism controls the orderly expression of genes during development while still permitting fine tuning in response to specific stimuli.

The neurodevelopmental disorder Rett Syndrome (RTT) is caused by sporadic mutations in the transcriptional factor methyl-CpG binding protein 2 (MeCP2). Although it is thought that the primary cause of RTT is cell autonomous due to lack of functional MeCP2 in neurons, whether non-cell autonomous factors contribute to the disease, is unknown. Here, we show that loss of MeCP2 occurs not only in neurons but also in glial cells of RTT brain. Using an in vitro co-culture system, we find that mutant astrocytes from a RTT mouse model, and their conditioned medium, fail to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. Our studies suggest that in RTT brain, astrocytes carrying MeCP2 mutations have a non-cell autonomous effect on neuronal properties, likely due to aberrant secretion of soluble factor(s).

Rett syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl CpG-binding protein 2 (MeCP2)1. Although MeCP2 is expressed in most tissues2, loss of MeCP2 results primarily in neurological symptoms1,3,4. Earlier studies propelled the idea that RTT is due exclusively to loss of MeCP2 function in neurons2,4-10. While defective neurons clearly underlie the aberrant behaviors, we and others showed recently that the loss of MeCP2 from glia negatively influences neurons in a non-cell autonomous fashion11-13. Here, we show that in globally MeCP2-deficient mice, re-expression of MeCP2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter (VGlut1). Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports targeting glia as a strategy for improving the associated symptoms.

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The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST͞NRSF (RE1 silencing transcription factor͞neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for A large number of genes encoding neuronal phenotypic traits, including ion channels, neurotransmitters, synaptic proteins, and cell-adhesion molecules, are expressed only in neurons. One mechanism important in establishing and maintaining this neural specificity involves the DNA-binding protein REST͞ NRSF (RE1 silencing transcription factor͞neural-restrictive silencing factor) (1-4), which serves to block expression of its target genes in nonneural tissues. Such maintained gene repression is in contrast to the more dynamic repression mechanism that regulates inducible gene expression in response to steroid hormone receptors, one of the best-studied mammalian repressor mechanisms (for review, see ref. 5).One REST target gene essential for neuronal physiology is that encoding the brain type II voltage-dependent sodium channel. This ion channel is required for the propagation of fast electrical signals in neurons, in the form of neuronal impulses, and is not expressed in nonneural tissues. As is true for other REST target genes, there is a reciprocal relationship between expression of the type II sodium channel gene and expression of REST. Additionally, when a REST expression plasmid is cotransfected into neuronal cells along with a type II sodium channel reporter, the expression of the reporter gene is reduced dramatically (1). This result indicates either that REST alone is sufficient to repress its target genes or that REST accessory factors are present in neuronal cells despite the absence of REST.Two distinct repressor domains have been identified and characterized in REST (6, 7). These domains are located in the amino and carboxyl termini of the protein. Both domains are required for full repression in the context of the intact molecule, but each domain is sufficient to repress type II sodium channel reporter genes when expressed as a Gal4 fusion protein (6). The C-terminal repressor domain contains a C 2 H 2 class zinc finger beginning approximately 40 aa upstream of the stop codon. Deleting this domain, or introducing a point mutation critical to the zinc finger motif, abolishes repressor activity (6). Because zinc finger motifs often mediate proteinprotein interactions, we proposed that REST might function in conjunction with other nuclear factors or corepressors.In this study, we find that repression of the type II sodium channel promoter by REST requires a newly identified protein, CoREST, which fulfills the criteria for a bona fide corepressor. CoREST is a repressor; mutations that disrupt CoREST...

Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett Syndrome (RTT), a severe autism spectrum disorder that affects about one in 10,000 female live births. Because mutations in the Mecp2 gene occur in the germ cells with onset of neurological symptoms occurring in early childhood, the role of MeCP2 has been ascribed to brain maturation at a specific developmental window. Here, we show similar kinetics of onset and progression of RTT-like symptoms in mice, including lethality, if MeCP2 is removed postnatally during the developmental stage that coincides with RTT onset, or adult stage. For the first time, we show that brains that lose MeCP2 at these two different stages are actively shrinking, resulting in higher than normal neuronal cell density. Furthermore, we show that mature dendritic arbors of pyramidal neurons are severely retracted and dendritic spine density is dramatically reduced. In addition, hippocampal astrocytes have significantly less complex ramified processes. These changes accompany a striking reduction in the levels of several synaptic proteins, including CaMKII α/β, AMPA and NMDA receptors, and the synaptic vesicle proteins Vglut and Synapsin, which represent critical modifiers of synaptic function and dendritic arbor structure. Importantly, the mRNA levels of these synaptic proteins remains unchanged, suggesting that MeCP2 likely regulates these synaptic proteins post-transcriptionally, directly or indirectly. Our data suggest a crucial role for MeCP2 in post-transcriptional regulation of critical synaptic proteins involved in maintaining mature neuronal networks during late stages of postnatal brain development.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a singlestranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5 end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAP␣, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAP␣ interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of At-KAP␣ was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin ␣ family. Indeed, AtKAP␣ efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAP␣ specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.