scholarly journals An E box mediates activation and repression of the acetylcholine receptor delta-subunit gene during myogenesis.

1993 ◽  
Vol 13 (9) ◽  
pp. 5133-5140 ◽  
Author(s):  
A M Simon ◽  
S J Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Development ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Growth Hormone Gene ◽  
Subunit Gene ◽  
Specific Expression ◽  
E Box ◽  
Nonmuscle Cells ◽  
Delta Subunit

The genes encoding the skeletal muscle acetylcholine receptor (AChR) are induced during muscle development and are regulated subsequently by innervation. Because both the initiation and the subsequent regulation of AChR expression are controlled by transcriptional mechanisms, an understanding of the steps that regulate AChR expression following innervation is likely to require knowledge of the pathway that activates AChR genes during myogenesis. Thus, we sought to identify the cis-acting sequences that regulate expression of the AChR delta-subunit gene during muscle differentiation. We transfected muscle and nonmuscle cell lines with gene fusions between 5'-flanking DNA from the AChR delta-subunit gene and the human growth hormone gene, and we show here that 148 bp of 5'-flanking DNA from the AChR delta-subunit gene contains two regulatory elements that control muscle-specific gene expression. One element is an E box, which is important both for activation of the delta-subunit gene in myotubes and for its repression in myoblasts and nonmuscle cells. Mutation of this E box, which prevents binding of MyoD-E2A and myogenin-E2A heterodimers, decreases expression in myotubes and increases expression in myoblasts and nonmuscle cells. An E-box binding activity, which does not contain MyoD, myogenin, or E2A proteins, is present in muscle and nonmuscle cells and may be responsible for repressing the delta-subunit gene in myoblasts and nonmuscle cells. An enhancer, which lacks E boxes, is also required for expression of the delta-subunit gene but does not confer muscle-specific expression.

An E box mediates activation and repression of the acetylcholine receptor delta-subunit gene during myogenesis

1993 ◽  
Vol 13 (9) ◽  
pp. 5133-5140
Author(s):  
A M Simon ◽  
S J Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Development ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Growth Hormone Gene ◽  
Subunit Gene ◽  
Specific Expression ◽  
E Box ◽  
Nonmuscle Cells ◽  
Delta Subunit

The genes encoding the skeletal muscle acetylcholine receptor (AChR) are induced during muscle development and are regulated subsequently by innervation. Because both the initiation and the subsequent regulation of AChR expression are controlled by transcriptional mechanisms, an understanding of the steps that regulate AChR expression following innervation is likely to require knowledge of the pathway that activates AChR genes during myogenesis. Thus, we sought to identify the cis-acting sequences that regulate expression of the AChR delta-subunit gene during muscle differentiation. We transfected muscle and nonmuscle cell lines with gene fusions between 5'-flanking DNA from the AChR delta-subunit gene and the human growth hormone gene, and we show here that 148 bp of 5'-flanking DNA from the AChR delta-subunit gene contains two regulatory elements that control muscle-specific gene expression. One element is an E box, which is important both for activation of the delta-subunit gene in myotubes and for its repression in myoblasts and nonmuscle cells. Mutation of this E box, which prevents binding of MyoD-E2A and myogenin-E2A heterodimers, decreases expression in myotubes and increases expression in myoblasts and nonmuscle cells. An E-box binding activity, which does not contain MyoD, myogenin, or E2A proteins, is present in muscle and nonmuscle cells and may be responsible for repressing the delta-subunit gene in myoblasts and nonmuscle cells. An enhancer, which lacks E boxes, is also required for expression of the delta-subunit gene but does not confer muscle-specific expression.

Separate pathways for synapse-specific and electrical activity-dependent gene expression in skeletal muscle

Development ◽  
1994 ◽  
Vol 120 (7) ◽  
pp. 1799-1804
Author(s):  
J. Tang ◽  
S.A. Jo ◽  
S.J. Burden
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Electrical Activity ◽  
Dna Sequences ◽  
Binding Protein ◽  
Specific Gene ◽  
Subunit Gene ◽  
E Box ◽  
Delta Subunit ◽  
Activity Dependent

Signaling between nerve and muscle is mediated by multiple mechanisms, including two transcriptional pathways. Signals provided by the nerve terminal activate transcription of acetylcholine receptor (AChR) genes in myofiber nuclei near the synaptic site, and signals associated with myofiber electrical activity inactivate AChR gene expression throughout the myofiber. These opposing effects of innervation are conferred by 1.8 kb of 5′ flanking DNA from the AChR delta subunit gene. These results raise the possibility that synapse-specific and electrical activity-dependent gene expression are mediated by the same DNA sequence and that activation and repression are determined by differential regulation of the same DNA binding protein. We produced transgenic mice carrying AChR delta subunit-hGH gene fusions, and we show here that a binding site (E-box) for myogenic basic helix-loop-helix proteins is required for electrical activity-dependent but not for synapse-specific gene expression of the delta subunit gene. These results indicate that a change in the expression or activity of an E-box binding protein(s) mediates electrical activity-dependent gene regulation and that synapse-specific and electrical activity-dependent gene expression require different DNA sequences. Moreover, we show here that the cis-acting elements for both aspects of innervation-dependent gene regulation are contained in 181 bp of 5′ flanking DNA from the AChR delta subunit gene.

Spatial restriction of AChR gene expression to subsynaptic nuclei

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 545-553 ◽  
Author(s):  
A.M. Simon ◽  
P. Hoppe ◽  
S.J. Burden
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Acetylcholine Receptors ◽  
Human Growth ◽  
Growth Hormone Gene ◽  
Subunit Gene ◽  
Specific Expression ◽  
Synaptic Region ◽  
Continuous Presence ◽  
Delta Subunit

Acetylcholine receptors (AChRs) and the mRNAs encoding the four AChR subunits are highly concentrated in the synaptic region of skeletal myofibers. The initial localization of AChRs to synaptic sites is triggered by the nerve and is caused, in part, by post-translational mechanisms that involve a redistribution of AChR protein in the myotube membrane. We have used transgenic mice that harbor a gene fusion between the murine AChR delta subunit gene and the human growth hormone gene to show that innervation also activates two independent transcriptional pathways that are important for establishing and maintaining this non-uniform distribution of AChR mRNA and protein. One pathway is triggered by signal(s) that are associated with myofiber depolarization, and these signals act to repress delta subunit gene expression in nuclei throughout the myofiber. Denervation of muscle removes this repression and causes activation of delta subunit gene expression in nuclei in non-synaptic regions of the myofiber. A second pathway is triggered by an unknown signal that is associated with the synaptic site, and this signal acts locally to activate delta subunit gene expression only in nuclei within the synaptic region. Synapse-specific expression, however, does not depend upon the continuous presence of the nerve, since transcriptional activation of the delta subunit gene in subsynaptic nuclei persists after denervation. Thus, the nuclei in the synaptic region of multinucleated skeletal myofibers are transcriptionally distinct from nuclei elsewhere in the myofiber, and this spatially restricted transcription pattern is presumably imposed initially by the nerve.

scholarly journals Genetic influences on cell type specific gene expression and splicing during neurogenesis elucidate regulatory mechanisms of brain traits

2020 ◽  
Author(s):  
Nil Aygün ◽  
Angela L. Elwell ◽  
Dan Liang ◽  
Michael J. Lafferty ◽  
Kerry E. Cheek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
Regulatory Mechanisms ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Risk Variants ◽  
Allele Specific ◽  
Cell Type Specific ◽  
Regulatory Effects

SummaryInterpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing is mainly performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements of cells present during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs and allele specific expression in primary human neural progenitors (n=85) and their sorted neuronal progeny (n=74). Using colocalization and TWAS, we uncover cell-type specific regulatory mechanisms underlying risk for these traits.

E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

scholarly journals Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

1996 ◽  
Vol 16 (6) ◽  
pp. 2777-2786 ◽  
Author(s):  
V Gailus-Durner ◽  
J Xie ◽  
C Chintamaneni ◽  
A K Vershon
Keyword(s):  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Expression ◽  
Autonomously Replicating Sequence

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

scholarly journals Stepwise activation of the mouse acetylcholine receptor delta- and gamma-subunit genes in clonal cell lines.

1988 ◽  
Vol 8 (12) ◽  
pp. 5257-5267 ◽  
Author(s):  
C M Crowder ◽  
J P Merlie
Keyword(s):  
Acetylcholine Receptor ◽  
Cell Lines ◽  
Subunit Gene ◽  
Rnase Protection ◽  
Gamma Subunit ◽  
Clonal Cell ◽  
Hypersensitive Sites ◽  
Clonal Cell Lines ◽  
Gamma Locus ◽  
Delta Subunit

We used the DNase I-hypersensitive sites around the mouse acetylcholine receptor delta-subunit gene as a guide toward the cloning and sequencing of delta and gamma transcriptional regulatory regions and as a means to assess chromatin structural activation of the delta- and gamma-subunit genes during myogenesis. Genomic cloning of hypersensitive sites downstream of the delta-subunit gene revealed the presence of the gamma-subunit gene approximately 5 kilobases away; the hypersensitive sites mapped to the 5' end of the gamma-subunit gene. Sequence comparison of restriction fragments containing hypersensitive sites in analogous locations at the 5' ends of the delta- and gamma-subunit genes uncovered little overall homology between the two genomic fragments; however, an 11- of 13-base-pair match between the two sequences was found. Homologs to this sequence were also found to be present in the upstream regions of the chicken alpha- and mouse beta-subunit genes. By RNase protection and primer extension analyses, the delta-subunit gene transcription start site was mapped to 56 base pairs upstream of the initiator ATG codon. Clonal cell lines with various potentials to differentiate to the skeletal muscle phenotype were examined for their hypersensitive site pattern within the delta-gamma locus. Only remote hypersensitive sites flanking the locus appear in pluripotential mesodermal cells. A cell line of determined but inducible myoblasts expressed only one more intergenic site, while in permissively differentiating myoblasts hypersensitive sites were already present at the 5' ends of the delta and gamma genes prior to differentiation. Terminal differentiation resulted in an identical pattern of hypersensitive sites in all muscle cell lines examined so far, with an intergenic site near the gamma-subunit gene being the only site specific to the differentiated muscle phenotype.

scholarly journals Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick.

1989 ◽  
Vol 108 (3) ◽  
pp. 1025-1037 ◽  
Author(s):  
B Fontaine ◽  
J P Changeux
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Muscle Development ◽  
Neuromuscular Junctions ◽  
Primary Cultures ◽  
Motor Endplate ◽  
Alpha Subunit ◽  
Subunit Gene ◽  
Nicotinic Acetylcholine

In 15-d-old chick latissimi dorsi muscles, the nicotinic acetylcholine receptor (AChR) alpha-subunit mRNA is densely accumulated at the level of subsynaptic nuclei of the motor endplate (Fontaine et al., 1988). In this paper, using in situ hybridization with genomic probes, we further show that the expression of the AChR alpha-subunit gene in the embryo, revealed by the accumulation of mature mRNAs, starts in myotomal cells and persists during the first stages of muscle development in a majority of muscle nuclei. Subsequently, the distribution of AChR alpha-subunit mRNAs becomes restricted to the newly formed motor endplates as neuromuscular junctions develop. To assess the transcriptional activity of individual nuclei in developing muscles, a strictly intronic fragment of the AChR alpha-subunit gene was used to probe in situ the level of unspliced transcripts. AChR alpha-subunit unspliced transcripts accumulate around a large number of sarcoplasmic nuclei at embryonic day 11, but can no longer be detected at their level after embryonic day 16 in the embryo. A similar decrease in the accumulation of AChR alpha-subunit transcripts is observed between day 4 and day 6 in primary cultures of muscle cells. On the other hand, in vivo denervation and in vitro blocking of muscle electrical activity by the sodium channel blocker tetrodotoxin results in an increase in the labeling of muscle nuclei. Yet, only 6% of the muscle nuclei appear labeled by the strictly intronic probes after denervation. The possible significance of such heterogeneity of muscle nuclei during motor endplate formation in AChR gene expression is discussed.

scholarly journals GC- and E-box Motifs as Regulatory Elements in the Proximal Promoter Region of the Neuronal Nicotinic Receptor α7 Subunit Gene

1998 ◽  
Vol 273 (32) ◽  
pp. 20021-20028 ◽  
Author(s):  
Carmen Carrasco-Serrano ◽  
Antonio Campos-Caro ◽  
Salvador Viniegra ◽  
Juan J. Ballesta ◽  
Manuel Criado
Keyword(s):  
Nicotinic Receptor ◽  
Promoter Region ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Subunit Gene ◽  
Proximal Promoter Region ◽  
Neuronal Nicotinic Receptor ◽  
E Box ◽  
Α7 Subunit

scholarly journals Isolation and characterization of the mouse acetylcholine receptor delta subunit gene: identification of a 148-bp cis-acting region that confers myotube-specific expression.

1988 ◽  
Vol 107 (6) ◽  
pp. 2271-2279 ◽  
Author(s):  
T J Baldwin ◽  
S J Burden
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Acetylcholine Receptor ◽  
Subunit Gene ◽  
Cell Type ◽  
Cis Acting ◽  
Mouse Skeletal Muscle ◽  
Cell Type Specific ◽  
Delta Subunit ◽  
Cat Gene

We have isolated the gene encoding the delta subunit of the mouse skeletal muscle acetylcholine receptor (AChR) and have identified a 148-bp cis-acting region that controls cell type-specific and differentiation-dependent gene expression. The 5' flanking region of the delta subunit gene was fused to the protein-coding region of the chloramphenicol acetyltransferase (CAT) gene and gene fusions were transfected into C2 mouse skeletal muscle cells. Both transiently and stably transfected cells were assayed for CAT gene expression. Deletions from the 5' end of the mouse delta gene demonstrate that 148 bp of 5' flanking DNA is sufficient to confer cell type-specific and differentiation-dependent expression: CAT activity is present in transfected myotubes, but not in transfected 3T3 cells or 10T1/2 cells. Moreover, the level of CAT expression in myotubes transfected with constructs containing 148 bp of 5' flanking DNA from the delta subunit gene is identical to that in myotubes transfected with constructs containing 3.2 kb of 5' flanking DNA and similar to expression from the SV-40 early promoter. Increased CAT activity in myotubes is a result of an increased rate of transcription from the delta subunit promoter, since CAT RNA levels are also 35-fold more abundant in myotubes than myoblasts. In contrast, the SV-40 early promoter is similarly active in all cell types. Thus, 148 bp of 5' flanking DNA from the delta subunit gene contains all the information required for cell type-specific and differentiation-dependent expression of the AChR delta subunit.

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