The basic helix-loop-helix protein, sharp-1, represses transcription by a histone deacetylase-dependent and histone deacetylase-independent mechanism.

Many aspects of neurogenesis and neuronal differentiation are controlled by basic helix-loop-helix (bHLH) proteins. One such factor is SHARP-1, initially identified on the basis of its sequence similarity to hairy. Unlike hairy, and atypically for bHLHs, SHARP-1 is expressed late in development, suggestive of a role in terminal aspects of differentiation. Nevertheless, the role of SHARP-1 and the identity of its target genes remain unknown. During the course of a one-hybrid screen for transcription factors that bind to regulatory domains of the M1 muscarinic acetylcholine receptor gene, we isolated the bHLH transcription factor SHARP-1. In this study, we investigated the functional role of SHARP-1 in regulating transcription. Fusion proteins of SHARP-1 tethered to the gal4 DNA binding domain repress both basal and activated transcription when recruited to either a TATA-containing or a TATAless promoter. Furthermore, we identified two independent repression domains that operate via distinct mechanisms. Repression by a domain in the C terminus is sensitive to the histone deacetylase inhibitor trichostatin A, whereas repression by the bHLH domain is insensitive to TSA. Furthermore, overexpression of SHARP-1 represses transcription from the M(1) promoter. This study represents the first report to assign a function to, and to identify a target gene for, the bHLH transcription factor SHARP-1.

Neurological disease: listening to gene silencers.

Neurons regulate the expression of genes essential to individual neuron function through elegant combinatorial interactions among a limited number of transcription factors. In addition, an economy of regulatory control is practiced within the nucleus that belies conceptual divisions of transcription factors into "repressors" and "activators." Studies of the neural restrictive silencer element (NRSE, also known as RE1) and its repressor protein have revealed a multitude of mechanisms by which transcriptional regulation is not only elaborated in normal neuronal development, but perverted in disease states.

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.

Repression and activation of muscarinic receptor genes.

The specific cellular response to muscarinic receptor activation is dependent upon appropriate expression of each of the five muscarinic receptor genes by individual cells. Here we summarise recent work describing some of the genomic regulatory elements and transcriptional mechanisms that control expression of the M1 and M4 genes.

Neural specific expression of the m4 muscarinic acetylcholine receptor gene is mediated by a RE1/NRSE-type silencing element.

Muscarinic receptor genes are members of the G-protein receptor superfamily that, with the inclusion of the odorant receptors, is believed to contain over a thousand members. Each member of this superfamily, which has been studied to date, appears to have a distinct pattern of expression, but little work has been done on the regulation of these complex expression patterns. We have recently isolated the rat m4 muscarinic receptor gene and identified a genomic 1520-nucleotide sequence that appeared capable of directing cell-specific expression (Wood, I. C., Roopra. A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940). In the present study we have constructed a set of deletion promoter constructs to more closely define the DNA elements that are responsible for m4 gene expression. We have found that deletion of a RE1/NRSE silencer element between nucleotides -574 and -550, similar to that found in other neural specific genes, results in activation of reporter expression in non-m4-expressing cells. Gel mobility shift analysis has shown that a protein present in nonexpressing cells is capable of binding to this element and is probably the recently identified neural silencer, REST/NRSF. Of the constitutively active proximal promoter only a tandem Sp-1 site appears to recruit DNA binding proteins that are present in all cells tested. This represents the first report documenting the role of this silencer in regulating expression of a member of the G-protein receptor family.

Structure of the m4 cholinergic muscarinic receptor gene and its promoter.

Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5'-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs (bp) upstream from the initiating ATG, separated from a 460-493 bp noncoding exon by a 4.8-kilobase intron. DNA sequence analysis shows that the non-coding exon is GC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elements including five Sp-1 binding sites, one AP-2 site, one AP-3 binding site and two E-boxes within the proximal 600 bp. A reporter construct consisting of 1440 bp of flanking DNA and 80 bp of the first exon cloned into a luciferase reporter plasmid, drove cell specific expression in transient transfection assays. Removal of 1088 bp of the 5' end of this construct resulted in expression in non-m4 expressing cell lines suggesting there is a repressor element in this region.

The construction and analysis of M13 libraries prepared from YAC DNA.

Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive. We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA. Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA. We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones.