Functional Properties of the Neuronal Nicotinic Acetylcholine Receptor β3 Promoter in the Developing Central Nervous System

Abstract

Within the chick central nervous system, expression of the ␤3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the ␤3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of ␤3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the ␤3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of ␤3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of ␤3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3 of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.In vertebrates, both negative and positive regulations play an important role in determining neuronal gene expression. Negative regulation (reviewed in Refs. 1 and 2) is best exemplified by REST/NRSF, a factor that represses transcription of the SCG-10 and type II Na ϩ channel genes in non-neuronal cell-types, whereas most neurons lack REST/NRSF and thus express these two genes (3, 4). Conversely, the Olf-1 transcription factor positively regulates several genes (e.g. the olfactory neuron-specific G protein, the type III adenylyl cyclase) specifically expressed in olfactory neurons (5). Vertebrate homologs of the basic helix-loop-helix (bHLH) 1 factors involved in Drosophila neurogenesis (6) act as positive or negative regulators in the acquisition of neuronal identity. For instance, the atonaland achaete-scute-related activators are transiently expressed in parts of the central and peripheral nervous system during early development, and their null mutation or ectopic expression profoundly influences neurogenesis (reviewed by Lee et al. (7)). However, the direct regulation by bHLH proteins of genes that define neuronal identity has never been documented.Several neuronal nicotinic acetylcholine receptor (nAChR) genes are expressed early in neural development (8 -12), and, since they encode transmembrane sensors capable of fluxing Ca 2ϩ and other cations upon stimulation (reviewed in Ref. 13), an understanding of their regulation should help explain how the genetic program puts together the mechanisms needed for epigenetic environmental cues to participate in development. This is illustrated in a recent report by Shatz and associates (14) showing that, in the developing retina, cholinergic synaptic transmission between newly generated amacrine and ganglion cells is respo...

Highly specific interactions between bHLH transcription factors and chromatin during retina development

“…A single E-box is implicated in the regulation of the β3 promoter (Fig. 1C) (Roztocil et al, 1998) and ATH5 was bound exclusively to sequences encompassing this region, but not to coding sequences located downstream of the transcription start site (ORF) (Fig. 2C).…”

The Basic Domain of ATH5 Mediates Neuron-Specific Promoter Activity during Retina Development

“…ATH5 is the only neuronal bHLH protein able to activate transcription of the ␤3 gene (22,31), and ChIP results demonstrate its in vivo binding to the ␤3 promoter in the early retina (33). Although it is coexpressed with ␤3 in newborn RGCs, NeuroM is unable to activate the ␤3 gene (23,31), and we wondered if that reflected an inability to bind the promoter during retina development. To address this question, we performed ChIP experiments using an antibody directed against NeuroM and chromatin prepared from E3 to E12 retinas and optic tectum.…”

“…Protein extractions and electromobility shift assays (EMSAs) were performed essentially as previously described (31). Bacterially expressed glutathione S-transferase (GST) fusion proteins were purified using glutathione beads according to the manufacturer's instructions (Amersham).…”

“…These residues are not involved in DNA binding but mediate interactions between ATH5 and chromatin. Previously, we had shown that ectopic expression of the myogenic factor MyoD stimulates expression of the ␤3 gene in central neurons that never express it in physiological conditions (31). One of the three amino acids conferring specificity on ATH5 is not conserved in the basic domain of MyoD, suggesting that MyoD activates ␤3 by a different mechanism.…”

“…ATH5 is directly involved in its own regulation and is able to activate genes, such as the ␤3 subunit of the neuronal nicotinic receptor and Brn3c, which specify neuronal identity traits (16,22,33). The relatively simple ␤3 promoter is sufficient to restrict reporter gene expression to RGCs (20) and is neither bound nor activated by neuronal bHLH transcription factors other than ATH5 present in the developing retina (31,33).We decided to determine how the ␤3 promoter selects ATH5 from among the other members of the bHLH protein family. ␤3 is coexpressed with ATH5 and NeuroM in newborn RGCs (23), and here we show that, whereas the ATH5 protein…”