A novel pathway for MuSK to induce key genes in neuromuscular synapse formation

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChRε . We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

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A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice

Molecular Endocrinology ◽

10.1210/mend.15.5.0628 ◽

2001 ◽

Vol 15 (5) ◽

pp. 734-746 ◽

Cited By ~ 3

Author(s):

Christine C. Quirk ◽

Kristen L. Lozada ◽

Ruth A. Keri ◽

John H. Nilson

Keyword(s):

Transgenic Mice ◽

Binding Site ◽

Cell Lines ◽

Regulatory Element ◽

Regulatory Elements ◽

Vital Role ◽

Proximal Region ◽

Expression Vectors ◽

Homeodomain Protein

Abstract Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

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Loading of DNA-Binding Factors to an Erythroid Enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.1993-2003.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 1993-2003 ◽

Cited By ~ 16

Author(s):

Shau-Ching Wen ◽

Karim Roder ◽

Kuang-Yu Hu ◽

Irene Rombel ◽

Narender R. Gavva ◽

...

Keyword(s):

Regulatory Element ◽

K562 Cells ◽

Globin Genes ◽

Erythroid Cells ◽

Binding Studies ◽

Mobility Shift ◽

Specific Expression ◽

Factor Binding ◽

Globin Promoter

ABSTRACT The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human α-like globin genes, the embryonic ζ and the adult α2/α/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3′-NA, modulates the capability of HS-40 to activate the embryonic ζ-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human α-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 1-bp, GC→TA mutation in the 3′-NA motif of HS-40, 3′-NA(II), has been shown previously to cause significant derepression of the embryonic ζ-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3′-NA motif. By gel mobility shift and transient cotransfection assays, we now show that 3′-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF-E2 as well as AP1 can still bind to the 3′-NA(II) sequence. The association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3′-NA motif. However, the 3′-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3′-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2–3′-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human α-globin locus via the 3′-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

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A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

Genome Biology ◽

10.1186/gb-2013-14-7-r72 ◽

2013 ◽

Vol 14 (7) ◽

pp. R72 ◽

Cited By ~ 11

Author(s):

Robin P Smith ◽

Samantha J Riesenfeld ◽

Alisha K Holloway ◽

Qiang Li ◽

Karl K Murphy ◽

...

Keyword(s):

Regulatory Element ◽

Specific Expression ◽

Tissue Specific ◽

Tissue Specific Expression ◽

In Vivo Screen

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PU.1/Pip and Basic Helix Loop Helix Zipper Transcription Factors Interact With Binding Sites in the CD20 Promoter to Help Confer Lineage- and Stage-Specific Expression of CD20 in B Lymphocytes

Blood ◽

10.1182/blood.v90.10.3984 ◽

1997 ◽

Vol 90 (10) ◽

pp. 3984-3995 ◽

Cited By ~ 60

Author(s):

Andreas Himmelmann ◽

Agostino Riva ◽

Gaye Lynn Wilson ◽

Brian P. Lucas ◽

Claire Thevenin ◽

...

Keyword(s):

B Cell ◽

Plasma Cells ◽

Expression Vectors ◽

Specific Gene ◽

Cell Stage ◽

Specific Expression ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box

Abstract CD20 is a B-lineage–specific gene expressed at the pre–B-cell stage of B-cell development that disappears on differentiation to plasma cells. As such, it serves as an excellent paradigm for the study of lineage and developmental stage-specific gene expression. Using in vivo footprinting we identified two sites in the promoter at −45 and −160 that were occupied only in CD20+ B cells. The −45 site is an E box that binds basic helix-loop-helix-zipper proteins whereas the −160 site is a composite PU.1 and Pip binding site. Transfection studies with reporter constructs and various expression vectors verified the importance of these sites. The composite PU.1 and Pip site likely accounts for both lineage and stage-specific expression of CD20 whereas the CD20 E box binding proteins enhance overall promoter activity and may link the promoter to a distant enhancer.

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Potential Autoregulation of Transcription Factor PU.1 by an Upstream Regulatory Element

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2832-2845.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2832-2845 ◽

Cited By ~ 114

Author(s):

Yutaka Okuno ◽

Gang Huang ◽

Frank Rosenbauer ◽

Erica K. Evans ◽

Hanna S. Radomska ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Reporter Gene ◽

Regulatory Element ◽

Mammalian Species ◽

Critical Role ◽

Homologous Region ◽

Specific Expression ◽

Upstream Regulatory Element

ABSTRACT Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb −14 site confers lineage-specific reporter gene expression in vivo. This kb −14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

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Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1220-1229.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1220-1229

Author(s):

K J Lee ◽

R Hickey ◽

H Zhu ◽

K R Chien

Keyword(s):

Transgenic Mice ◽

Myosin Light Chain ◽

Regulatory Element ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Specific Expression ◽

Negative Regulatory Element ◽

Reporter Activity ◽

E Box

The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue, HF-3 appears to function as a novel negative regulatory element to primarily suppress expression in muscle tissues. Thus, a combination of positive (HF-1a/HF-1b) and negative (E-box and HF-3) regulatory elements appear to be required to maintain ventricular chamber-specific expression in the in vivo context.

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GATA Motif on the Erythropoietin Gene Promoter Is Essential for Repression of Ectopic Constitutive Erythropoietin Production.

Blood ◽

10.1182/blood.v106.11.3139.3139 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3139-3139

Author(s):

Naoshi Obara ◽

Norio Suzuki ◽

Kim Ki-Bom ◽

Shigehiko Imagawa ◽

Toshiro Nagasawa ◽

...

Keyword(s):

Gene Expression ◽

Fluorescent Protein ◽

Regulatory Element ◽

Ectopic Expression ◽

Central Vein ◽

Specific Expression ◽

Neuronal Markers ◽

Nucleotide Mutation ◽

Gata Motif

Abstract In response to anemia erythropoietin ( Epo) gene transcription is markedly induced in the kidney and liver. This regulation has been ascribed for the promoter GATA motif and 3′ enhancer. To elucidate how the Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of renal cortex and hepatocytes surrounding the central vein. Surprisingly, the renal Epo-producing cells were identified with neuron-like morphology and expression of neuronal markers. We also examined the regulatory mechanism of Epo gene using the transgenes in which mutations had been introduced in the GATA promoter motif, since we have reported the motif as a negative regulatory element for the inducible Epo gene expression. A single nucleotide mutation in the motif resulted in constitutive ectopic expression of GFP in the renal distal tubules, collecting ducts and certain epithelial cells of other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in the distal tubular cells, these factors are likely to constitutively repress ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell-type specific expression of the Epo gene.

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APP interacts with LRP4 and agrin to coordinate the development of the neuromuscular junction in mice

eLife ◽

10.7554/elife.00220 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 40

Author(s):

Hong Y Choi ◽

Yun Liu ◽

Christian Tennert ◽

Yoshie Sugiura ◽

Andromachi Karakatsani ◽

...

Keyword(s):

Neuromuscular Junction ◽

Molecular Mechanisms ◽

Synapse Formation ◽

Transmembrane Domain ◽

Neuromuscular Synapse ◽

Apoe Receptor ◽

Cultured Myotubes ◽

Perinatal Lethality ◽

And Function ◽

Achr Clustering

ApoE, ApoE receptors and APP cooperate in the pathogenesis of Alzheimer’s disease. Intriguingly, the ApoE receptor LRP4 and APP are also required for normal formation and function of the neuromuscular junction (NMJ). In this study, we show that APP interacts with LRP4, an obligate co-receptor for muscle-specific tyrosine kinase (MuSK). Agrin, a ligand for LRP4, also binds to APP and co-operatively enhances the interaction of APP with LRP4. In cultured myotubes, APP synergistically increases agrin-induced acetylcholine receptor (AChR) clustering. Deletion of the transmembrane domain of LRP4 (LRP4 ECD) results in growth retardation of the NMJ, and these defects are markedly enhanced in APP−/−;LRP4ECD/ECD mice. Double mutant NMJs are significantly reduced in size and number, resulting in perinatal lethality. Our findings reveal novel roles for APP in regulating neuromuscular synapse formation through hetero-oligomeric interaction with LRP4 and agrin and thereby provide new insights into the molecular mechanisms that govern NMJ formation and maintenance.

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Repression of c-Kit and Its Downstream Substrates by GATA-1 Inhibits Cell Proliferation during Erythroid Maturation

Molecular and Cellular Biology ◽

10.1128/mcb.25.15.6747-6759.2005 ◽

2005 ◽

Vol 25 (15) ◽

pp. 6747-6759 ◽

Cited By ~ 76

Author(s):

Veerendra Munugalavadla ◽

Louis C. Dore ◽

Bai Lin Tan ◽

Li Hong ◽

Melanie Vishnu ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Regulatory Element ◽

Cytokine Signaling ◽

Cycle Arrest ◽

Erythroid Maturation ◽

Signaling Components

ABSTRACT Stem cell factor (SCF), erythropoietin (Epo), and GATA-1 play an essential role(s) in erythroid development. We examined how these proteins interact functionally in G1E cells, a GATA-1− erythroblast line that proliferates in an SCF-dependent fashion and, upon restoration of GATA-1 function, undergoes GATA-1 proliferation arrest and Epo-dependent terminal maturation. We show that SCF-induced cell cycle progression is mediated via activation of the Src kinase/c-Myc pathway. Restoration of GATA-1 activity induced G1 cell cycle arrest coincident with repression of c-Kit and its downstream effectors Vav1, Rac1, and Akt. Sustained expression of each of these individual signaling components inhibited GATA-1-induced cell cycle arrest to various degrees but had no effects on the expression of GATA-1-regulated erythroid maturation markers. Chromatin immunoprecipitation analysis revealed that GATA-1 occupies a defined Kit gene regulatory element in vivo, suggesting a direct mechanism for gene repression. Hence, in addition to its well-established function as an activator of erythroid genes, GATA-1 also participates in a distinct genetic program that inhibits cell proliferation by repressing the expression of multiple components of the c-Kit signaling axis. Our findings reveal a novel aspect of molecular cross talk between essential transcriptional and cytokine signaling components of hematopoietic development.

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Automated in vivo drug screen in zebrafish identifies synapse-stabilising drugs with relevance to spinal muscular atrophy

Disease Models & Mechanisms ◽

10.1242/dmm.047761 ◽

2021 ◽

Vol 14 (4) ◽

Author(s):

Ana-Maria Oprişoreanu ◽

Hannah L. Smith ◽

Sophia Krix ◽

Helena Chaytow ◽

Neil O. Carragher ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Small Molecule ◽

Synapse Formation ◽

Muscular Atrophy ◽

Neuromuscular Synapse ◽

Axon Growth ◽

Zebrafish Model ◽

Synaptic Site ◽

Automated Screening

ABSTRACT Synapses are particularly vulnerable in many neurodegenerative diseases and often the first to degenerate, for example in the motor neuron disease spinal muscular atrophy (SMA). Compounds that can counteract synaptic destabilisation are rare. Here, we describe an automated screening paradigm in zebrafish for small-molecule compounds that stabilize the neuromuscular synapse in vivo. We make use of a mutant for the axonal C-type lectin chondrolectin (chodl), one of the main genes dysregulated in SMA. In chodl−/− mutants, neuromuscular synapses that are formed at the first synaptic site by growing axons are not fully mature, causing axons to stall, thereby impeding further axon growth beyond that synaptic site. This makes axon length a convenient read-out for synapse stability. We screened 982 small-molecule compounds in chodl chodl−/− mutants and found four that strongly rescued motor axon length. Aberrant presynaptic neuromuscular synapse morphology was also corrected. The most-effective compound, the adenosine uptake inhibitor drug dipyridamole, also rescued axon growth defects in the UBA1-dependent zebrafish model of SMA. Hence, we describe an automated screening pipeline that can detect compounds with relevance to SMA. This versatile platform can be used for drug and genetic screens, with wider relevance to synapse formation and stabilisation.

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