scholarly journals Regulation of the Rapsyn Promoter by Kaiso and δ-Catenin

2004 ◽  
Vol 24 (16) ◽  
pp. 7188-7196 ◽  
Author(s):  
Marianna Rodova ◽  
Kevin F. Kelly ◽  
Michael VanSaun ◽  
Juliet M. Daniel ◽  
Michael J. Werle
Keyword(s):  
Transcription Factor ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Consensus Sequence ◽  
Specific Protein ◽  
C2c12 Cells ◽  
Congenital Myasthenic Syndrome ◽  
P120 Catenin ◽  
Myasthenic Syndrome

ABSTRACT Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that δ-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and δ-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and δ-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and δ-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, δ-catenin, and myogenic transcription factors at the neuromuscular junction.

scholarly journals Processing of ARIA and release from isolated nerve terminals

1999 ◽  
Vol 354 (1381) ◽  
pp. 411-416 ◽  
Author(s):  
Bomie Han ◽  
Gerald D. Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Action Potential ◽  
Acetylcholine Receptors ◽  
Molecular Layer ◽  
Postsynaptic Membrane ◽  
High Density ◽  
Small Contribution ◽  
Presynaptic Nerve Terminal ◽  
Voltage Gated

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage–gated Na + channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage–gated Na + channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo . ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.

scholarly journals Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 1938-1947 ◽  
Author(s):  
Tomohiko Tamura ◽  
Pratima Thotakura ◽  
Tetsuya S. Tanaka ◽  
Minoru S. H. Ko ◽  
Keiko Ozato
Keyword(s):  
Transcription Factor ◽  
Cystatin C ◽  
Target Genes ◽  
De Novo ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Microarray Gene Expression ◽  
Cathepsin C ◽  
Cis Element

Abstract Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8–induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

scholarly journals In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

1975 ◽  
Vol 66 (1) ◽  
pp. 209-213 ◽  
Author(s):  
H C Fertuck ◽  
W Woodward ◽  
M M Salpeter
Keyword(s):  
Fine Structure ◽  
Neuromuscular Junction ◽  
Muscle Contraction ◽  
Acetylcholine Receptors ◽  
Actinomycin D ◽  
Recovery Period ◽  
Em Autoradiography ◽  
Alpha Bungarotoxin ◽  
In Vivo Recovery

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.

scholarly journals The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

2000 ◽  
Vol 116 (3) ◽  
pp. 327-340 ◽  
Author(s):  
Claudio Grosman ◽  
Frank N. Salamone ◽  
Steven M. Sine ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Control Element ◽  
Dependent Manner ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Channel Opening ◽  
Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

scholarly journals Intrafamilial variation in clinical manifestations and response to salbutamol in siblings with congenital myasthenic syndrome caused by DOK7 mutations

2019 ◽  
Author(s):  
Trupti Jadhav ◽  
Poornima Shah ◽  
Purva Keni Karnavat ◽  
Anaita Udwadia Hegde
Keyword(s):  
Neuromuscular Junction ◽  
Early Life ◽  
Conventional Treatment ◽  
Clinical Manifestations ◽  
Respiratory Muscles ◽  
Congenital Myasthenic Syndrome ◽  
Late Childhood ◽  
Genetic Defects ◽  
Myasthenic Syndrome ◽  
Myasthenic Syndromes

The congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders arising from genetic defects in presynaptic, synaptic, and postsynaptic proteins of the neuromuscular junction (NMJ) resulting in variable and characteristically fatigable muscle weakness affecting limb, ocular, bulbar, trunk, and respiratory muscles from early life. DOK7 mutation resulting in synaptic and postsynaptic CMS clinically presents with limb-girdle myasthenia with sparing of facial and EOM. They characteristically worsen with conventional treatment and show excellent response to salbutamol/ ephedrine. Here we present a case highlighting a varied presentation beginning in late childhood and its evolution to reveal its congenital nature and subsequent management with salbutamol.

scholarly journals Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice. Running title: Tcfl5 is haploinsufficient in vivo

2021 ◽  
Author(s):  
Weiya Xu ◽  
Yiyun Zhang ◽  
Dongdong Qin ◽  
Yiqian Gui ◽  
Shu Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Specific Protein ◽  
Dual Function ◽  
Specific Cell ◽  
Chip Sequencing ◽  
Dna And Rna

Tissue-specific transcription factors often play key roles in the development of specific cell lineages. Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, although the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did, however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally regulated a set of genes participating in male germ cell development, which we uncovered via RNA-sequencing and TCFL5 ChIP-sequencing. We also found that TCFL5 interacted with RNA-binding proteins (RBPs) that regulated RNA processing, and further identified the fragile X mental retardation gene 1, autosomal homolog (FXR1, a known RBP) as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and hypothesize that TCFL5 may be a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

scholarly journals Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C Branon ◽  
Alice Y Ting ◽  
Dominique C Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

H1TF2A, the large subunit of a heterodimeric, glutamine-rich CCAAT-binding transcription factor involved in histone H1 cell cycle regulation

1994 ◽  
Vol 14 (12) ◽  
pp. 8322-8332
Author(s):  
R Martinelli ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Large Subunit ◽  
Consensus Sequence ◽  
Histone H1 ◽  
Technical Problems ◽  
Temporal Regulation ◽  
Cold Spring ◽  
Cycle Regulation

H1TF2 is a CCAAT transcription factor that binds to the histone H1 subtype-specific consensus sequence, which has previously been shown to be necessary for temporal regulation of histone H1 transcription during the cell cycle (F. La Bella, P. Gallinari, J. McKinney, and N. Heintz, Genes Dev. 3:1982-1990, 1989). In this study, we report that H1TF2 is a heteromeric CCAAT-binding protein composed of two polypeptide doublets of 33 and 34 kDa and 43 and 44 kDa that are not antigenically related. The 33- and 34-kDa species were not detected in our previous studies (P. Gallinari, F. La Bella, and N. Heintz, Mol. Cell. Biol. 9:1566-1575, 1989) because of technical problems in detection of these heavily glycosylated subunits. The cloning of H1TF2A, the large subunit of this factor, reveals it to be a glutamine-rich protein with extremely limited similarity to previously cloned CCAAT-binding proteins. A monospecific antiserum produced against bacterially synthesized H1TF2A was used to establish that HeLa cell H1TF2A is phosphorylated in vivo and that, in contrast to the H2b transcription factor Oct1 (S. B. Roberts, N. Segil, and N. Heintz, Science 253:1022-1026, 1991; N. Segil, S. B. Roberts, and N. Heintz, Cold Spring Harbor Symp. Quant. Biol. 56:285-292, 1991), no gross change in H1TF2A phosphorylation is evident during the cell cycle. Further immunoprecipitation studies demonstrated that H1TF2 is heterodimeric in the absence of DNA in vivo and identified several H1TF2-interacting proteins that may play a role in H1TF2 function in vivo.

scholarly journals Activity-Dependent Regulation of the Binomial Parameters p and n at the Mouse Neuromuscular Junction In Vivo

2010 ◽  
Vol 104 (5) ◽  
pp. 2352-2358 ◽  
Author(s):  
Xueyong Wang ◽  
Qingbo Wang ◽  
Kathrin L. Engisch ◽  
Mark M. Rich
Keyword(s):  
Neuromuscular Junction ◽  
Muscle Activity ◽  
Acetylcholine Receptors ◽  
Quantal Content ◽  
Spontaneous Release ◽  
Postsynaptic Activity ◽  
Activity Dependent

Block of neurotransmission at the mammalian neuromuscular junction triggers an increase in the number of vesicles released (quantal content). The increase occurs whether nerve and muscle activity are both blocked by placement of a tetrodotoxin (TTX) containing cuff on the nerve or whether muscle activity is selectively blocked by injection of α-bungarotoxin (BTX). We used ANOVA to examine whether the mechanism underlying the increase in quantal content differed between the two types of activity blockade. We found that TTX-induced blockade increased the probability of release ( p), whereas BTX-induced blockade increased the number of releasable vesicles ( n). The lack of increase in p when postsynaptic activity was blocked with BTX suggests that block of presynaptic activity triggers the increase. To determine whether n is regulated by mismatch of pre- and postsynaptic activity introduced by BTX injection we combined BTX and TTX and still found an increase in n. We conclude that block of acetylcholine binding to acetylcholine receptors during spontaneous release triggers the increase in n.

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