Axis formation and polarity in plants.

We have recently gained insight into a number of mechanisms governing the formation of the major axes that define the embryonic and adult plant body plan. Phenotypic analysis and molecular characterization of mutants with aberrant morphogenesis has led to a better understanding of key processes including the generation of the shape of the apical embryo, the establishment and maintenance of the radial pattern of the root, and the placement of lateral organ primordia around the shoot apical meristem.

Constitutive expression of the neuron-restrictive silencer factor (NRSF)/REST in differentiating neurons disrupts neuronal gene expression and causes axon pathfinding errors in vivo.

The neuron-restrictive silencer factor (NRSF; also known as REST for repressor element-1 silencing transcription factor) is a transcriptional repressor of multiple neuronal genes, but little is known about its function in vivo. NRSF is normally down-regulated upon neuronal differentiation. Constitutive expression of NRSF in the developing spinal cord of chicken embryos caused repression of two endogenous target genes, N-tubulin and Ng-CAM, but did not prevent overt neurogenesis. Nevertheless, commissural neurons that differentiated while constitutively expressing NRSF showed a significantly increased frequency of axon guidance errors. These data suggest that down-regulation of NRSF is necessary for the proper development of at least some classes of neurons in vivo.

Identification of a cell type-specific silencer in the first exon of the His-1 gene.

The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from -335 to -168 and -617 to -335, respectively. A strong His-1-positive regulatory element (HPRE, +18 to +29) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or SV40 promoters by greater than 80%. This negative regulatory sequence interacted with nuclear proteins from cells that contain a silent His-1 gene but showed no interaction with nuclear proteins from cells that actively transcribe the endogenous gene. HNRE-mediated repression was orientation independent; most of this activity was mapped to a minimal 26-bp sequence. These findings suggest that the first exon of the His-1 gene contains a cell type-specific silencer that contributes to the regulation of His-1 transcription.

Transcriptional repression by neuron-restrictive silencer factor is mediated via the Sin3-histone deacetylase complex.

A large number of neuron-specific genes characterized to date are under the control of negative transcriptional regulation. Many promoter regions of neuron-specific genes possess the repressor element repressor element 1/neuron-restrictive silencing element (RE1/NRSE). Its cognate binding protein, REST/NRSF, is an essential transcription factor; its null mutations result in embryonic lethality, and its dominant negative mutants produce aberrant expression of neuron-specific genes. REST/NRSF acts as a regulator of neuron-specific gene expression in both nonneuronal tissue and developing neurons. Here, we shown that heterologous expression of REST/NRSF in Saccharomyces cerevisiae is able to repress transcription from yeast promoters engineered to contain RE1/NRSEs. Moreover, we have taken advantage of this observation to show that this repression requires both yeast Sin3p and Rpd3p and that REST/NRSF physically interacts with the product of the yeast SIN3 gene in vivo. Furthermore, we show that REST/NRSF binds mammalian SIN3A and HDAC-2 and requires histone deacetylase activity to repress neuronal gene transcription in both nonneuronal and neuronal cell lines. We show that REST/NRSF binding to RE1/NRSE is accompanied by a decrease in the acetylation of histones around RE1/NRSE and that this decrease requires the N-terminal Sin3p binding domain of REST/NRSF. Taken together, these data suggest that REST/NRSF represses neuronal gene transcription by recruiting the SIN3/HDAC complex.

Protein kinase A regulates cholinergic gene expression in PC12 cells: REST4 silences the silencing activity of neuron-restrictive silencer factor/REST.

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A-deficient PC12 mutant, A126.1B2, in which transcription of the gene is reduced. The site of action of protein kinase A was localized to a neuron-restrictive silencer element/repressor element 1 (NRSE/RE-1) sequence within the cholinergic gene. Neuron-restrictive silencer factor (NRSF)/RE-1-silencing transcription factor (REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2 cells. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform REST4 was expressed in PC12 cells but not in A126.1B2. REST4 inhibited binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Coimmunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in A126.1B2 was sufficient to transcriptionally activate the cholinergic gene locus. Thus, in PC12 cells, protein kinase A promotes the production of REST4, which inhibits repression of the cholinergic gene locus by NRSF/REST.

NRSF/REST is required in vivo for repression of multiple neuronal target genes during embryogenesis.

The neuron-restrictive silencer factor NRSF (also known as REST and XBR) can silence transcription from neuronal promoters in non-neuronal cell lines, but its function during normal development is unknown. In mice, a targeted mutation of Rest, the gene encoding NRSF, caused derepression of neuron-specific tubulin in a subset of non-neural tissues and embryonic lethality. Mosaic inhibition of NRSF in chicken embryos, using a dominant-negative form of NRSF, also caused derepression of neuronal tubulin, as well as of several other neuronal target genes, in both non-neural tissues and central nervous system neuronal progenitors. These results indicate that NRSF is required to repress neuronal gene expression in vivo, in both extra-neural and undifferentiated neural tissue.

Identification of potential target genes for the neuron-restrictive silencer factor.

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.

Regulation of Pax-3 expression in the dermomyotome and its role in muscle development.

The segmented mesoderm in vertebrates gives rise to a variety of cell types in the embryo including the axial skeleton and muscle. A number of transcription factors containing a paired domain (Pax proteins) are expressed in the segmented mesoderm during embryogenesis. These include Pax-3 and a closely related gene, Pax-7, both of which are expressed in the segmental plate and in the dermomyotome. In this paper, we show that signals from the notochord pattern the expression of Pax-3, Pax-7 and Pax-9 in somites and the subsequent differentiation of cell types that arise from the somitic mesoderm. We directly assess the role of the Pax-3 gene in the differentiation of cell types derived from the dermomyotome by analyzing the development of muscle in splotch mouse embryos which lack a functional Pax-3 gene. A population of Pax-3-expressing cells derived from the dermomyotome that normally migrate into the limb are absent in homozygous splotch embryos and, as a result, limb muscles are lost. No abnormalities were detected in the trunk musculature of splotch embryos indicating that Pax-3 is necessary for the development of the limb but not trunk muscle.