scholarly journals Physical interactions between Gsx2 and Ascl1 regulate the balance between progenitor expansion and neurogenesis in the mouse lateral ganglionic eminence

2019 ◽  
Author(s):  
Kaushik Roychoudhury ◽  
Joseph Salomone ◽  
Shenyue Qin ◽  
Masato Nakafuku ◽  
Brian Gebelein ◽  
...  
Keyword(s):  
Dna Binding ◽  
Ventricular Zone ◽  
Luciferase Assay ◽  
Proximity Ligation Assay ◽  
Ganglionic Eminence ◽  
Independent Manner ◽  
Helix Loop Helix ◽  
Lateral Ganglionic Eminence ◽  
Physical Interactions

AbstractThe Gsx2 homeodomain transcription factor is required to maintain neural progenitor identity in the lateral ganglionic eminence (LGE) within the developing ventral telencephalon, despite its role in upregulating the neurogenic factor Ascl1. How Gsx2 maintains cells as progenitors in the presence of a pro-differentiation factor is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in dividing subapical progenitors within the LGE ventricular zone (VZ). Moreover, we show that while Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors that do not express Gsx2, co-expression of Gsx2 with Ascl1 inhibits neurogenesis in these cells. To investigate the mechanisms underlying this inhibition, we used a cell-based luciferase assay to show that Gsx2 reduced the ability of Ascl1 to activate target gene expression in a dose-dependent and DNA binding-independent manner. Yeast 2-hybrid and co-immunoprecipitation assays revealed that Gsx2 physically interacts with the basic-Helix-Loop-Helix (bHLH) domain of Ascl1, and DNA binding assays demonstrated that this interaction interferes with the ability of Ascl1 to form homo- or heterodimers with E-proteins such as Tcf3 on DNA. To further assess for in vivo molecular interactions between these transcription factors within the telencephalon, we modified a proximity ligation assay for embryonic tissue sections and found that Ascl1:Gsx2 interactions are enriched within VZ progenitors, whereas Ascl1:Tcf3 interactions predominate in basal progenitors. Altogether, these findings suggest that physical interactions between Gsx2 and Ascl1 limit Ascl1:Ascl1 and Ascl1:Tcf3 interactions, and thereby inhibit Ascl1-dependennt neurogenesis and allow for progenitor expansion within the LGE.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

NeuroM, a neural helix-loop-helix transcription factor, defines a new transition stage in neurogenesis

Development ◽  
1997 ◽  
Vol 124 (17) ◽  
pp. 3263-3272 ◽  
Author(s):  
T. Roztocil ◽  
L. Matter-Sadzinski ◽  
C. Alliod ◽  
M. Ballivet ◽  
J.M. Matter
Keyword(s):  
Nervous System ◽  
Transcription Factor ◽  
Mitotic Cycle ◽  
Ventricular Zone ◽  
Helix Loop Helix ◽  
E Box ◽  
Genes Encoding ◽  
Developing Nervous System

Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.

scholarly journals Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer

2012 ◽  
Vol 68 (11) ◽  
pp. 1354-1358 ◽  
Author(s):  
Marie V. Wong ◽  
Paaventhan Palasingam ◽  
Prasanna R. Kolatkar
Keyword(s):  
Dna Binding ◽  
Potassium Acetate ◽  
Class Iii ◽  
Data Set ◽  
Cell Parameters ◽  
Helix Loop Helix ◽  
Id Protein ◽  
Bhlh Transcription Factors

The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix–loop–helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-livesin vivoand reportedin vitroinstability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1 Å resolution data set was collected from a crystal belonging to space groupP3121 with unit-cell parametersa=b= 51.622,c= 111.474 Å, α = β = 90, γ = 120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1 MMES pH 6.5, 2.0 Mpotassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.

scholarly journals The nucleoid as a scaffold for the assembly of bacterial signaling complexes

2017 ◽  
Author(s):  
Audrey Moine ◽  
Leon Espinosa ◽  
Eugenie Martineau ◽  
Mutum Yaikhomba ◽  
P J Jazleena ◽  
...  
Keyword(s):  
Dna Binding ◽  
Independent Manner ◽  
Signaling Systems ◽  
Bacterial Signaling ◽  
Aminoacid Sequence ◽  
Membrane Bound ◽  
Gliding Bacterium ◽  
Positively Charged

ABSTRACTThe FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.AUTHOR SUMMARYIn this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the Frz kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

scholarly journals Physical interactions between Gsx2 and Ascl1 balance progenitor expansion versus neurogenesis in the mouse lateral ganglionic eminence

Development ◽  
2020 ◽  
Vol 147 (7) ◽  
pp. dev185348 ◽  
Author(s):  
Kaushik Roychoudhury ◽  
Joseph Salomone ◽  
Shenyue Qin ◽  
Brittany Cain ◽  
Mike Adam ◽  
...  
Keyword(s):  
Ganglionic Eminence ◽  
Lateral Ganglionic Eminence ◽  
Physical Interactions

scholarly journals Sequential XylS-CTD Binding to the Pm Promoter Induces DNA Bending Prior to Activation

2010 ◽  
Vol 192 (11) ◽  
pp. 2682-2690 ◽  
Author(s):  
Patricia Domínguez-Cuevas ◽  
Juan-Luís Ramos ◽  
Silvia Marqués
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Independent Manner ◽  
Form Complex ◽  
Terminal Domain ◽  
Proximal Site ◽  
Footprint Analysis ◽  
Global Curvature ◽  
Polymerase Binding

ABSTRACT XylS protein, a member of the AraC family of transcriptional regulators, comprises a C-terminal domain (CTD) involved in DNA binding and an N-terminal domain required for effector binding and protein dimerization. In the absence of benzoate effectors, the N-terminal domain behaves as an intramolecular repressor of the DNA binding domain. To date, the poor solubility properties of the full-length protein have restricted XylS analysis to genetic approaches in vivo. To characterize the molecular consequences of XylS binding to its operator, we used a recombinant XylS-CTD variant devoid of the N-terminal domain. The resulting protein was soluble and monomeric in solution and activated transcription from its cognate promoter in an effector-independent manner. XylS binding sites in the Pm promoter present an intrinsic curvature of 35° centered at position −42 within the proximal site. Gel retardation and DNase footprint analysis showed XylS-CTD binding to Pm occurred sequentially: first a XylS-CTD monomer binds to the proximal site overlapping the RNA polymerase binding sequence to form complex I. This first event increased Pm bending to 50° and was followed by the binding of the second monomer, which further increased the observed global curvature to 98°. This generated a concomitant shift in the bending center to a region centered at position −51 when the two sites were occupied (complex II). We propose a model in which DNA structure and binding sequences strongly influence XylS binding events previous to transcription activation.

scholarly journals The Basic Helix-Loop-Helix Transcription Factor NeuroD1 Facilitates Interaction of Sp1 with the Secretin Gene Enhancer

2007 ◽  
Vol 27 (22) ◽  
pp. 7839-7847 ◽  
Author(s):  
Subir K. Ray ◽  
Andrew B. Leiter
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Target Genes ◽  
Zinc Fingers ◽  
Homeodomain Proteins ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Gene Enhancer ◽  
Genes Encoding

ABSTRACT The basic helix-loop-helix transcription factor NeuroD1 is required for late events in neuronal differentiation, for maturation of pancreatic β cells, and for terminal differentiation of enteroendocrine cells expressing the hormone secretin. NeuroD1-null mice demonstrated that this protein is essential for expression of the secretin gene in the murine intestine, and yet it is a relatively weak transcriptional activator by itself. The present study shows that Sp1 and NeuroD1 synergistically activate transcription of the secretin gene. NeuroD1, but not its widely expressed dimerization partner E12, physically interacts with the C-terminal 167 amino acids of Sp1, which include its DNA binding zinc fingers. NeuroD1 stabilizes Sp1 DNA binding to an adjacent Sp1 binding site on the promoter to generate a higher-order DNA-protein complex containing both proteins and facilitates Sp1 occupancy of the secretin promoter in vivo. NeuroD-dependent transcription of the genes encoding the hormones insulin and proopiomelanocortin is potentiated by lineage-specific homeodomain proteins. The stabilization of binding of the widely expressed transcription factor Sp1 to the secretin promoter by NeuroD represents a distinct mechanism from other NeuroD target genes for increasing NeuroD-dependent transcription.

scholarly journals Pf1, a Novel PHD Zinc Finger Protein That Links the TLE Corepressor to the mSin3A-Histone Deacetylase Complex

2001 ◽  
Vol 21 (13) ◽  
pp. 4110-4118 ◽  
Author(s):  
Gregory S. Yochum ◽  
Donald E. Ayer
Keyword(s):  
Dna Binding ◽  
Histone Deacetylase ◽  
Zinc Finger Protein ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
Binding Domain ◽  
Independent Manner ◽  
Alpha Helices ◽  
Interacting Protein

ABSTRACT The mSin3A-histone deacetylase corepressor is a multiprotein complex that is recruited by DNA binding transcriptional repressors. Sin3 has four paired amphipathic alpha helices (PAH1 to -4) that are protein-protein interaction motifs and is the scaffold upon which the complex assembles. We identified a novel mSin3A-interacting protein that has two plant homeodomain (PHD) zinc fingers we term Pf1, for PHD factor one. Pf1 associates with mSin3A in vivo and recruits the mSin3A complex to repress transcription when fused to the DNA binding domain of Gal4. Pf1 interacts with Sin3 through two independent Sin3 interaction domains (SIDs), Pf1SID1 and Pf1SID2. Pf1SID1 binds PAH2, while Pf1SID2 binds PAH1. Pf1SID1 has sequence and structural similarity to the well-characterized 13-amino-acid SID of the Mad bHLHZip repressor. Pf1SID2 does not have sequence similarity with either Mad SID or Pf1SID1 and therefore represents a novel Sin3 binding domain. Mutations in a minimal fragment of Pf1 that encompasses Pf1SID1 inhibited mSin3A binding yet only slightly impaired repression when targeted to DNA, implying that Pf1 might interact with other corepressors. We show that Pf1 interacts with a mammalian homolog of the Drosophila Groucho corepressor, transducin-like enhancer (TLE). Pf1 binds TLE in an mSin3A-independent manner and recruits functional TLE complexes to repress transcription. These findings suggest that Pf1 may serve to bridge two global transcription networks, mSin3A and TLE.

scholarly journals Id Helix-Loop-Helix Proteins Antagonize Pax Transcription Factor Activity by Inhibiting DNA Binding

2001 ◽  
Vol 21 (2) ◽  
pp. 524-533 ◽  
Author(s):  
E. Claire Roberts ◽  
Richard W. Deed ◽  
Toshiaki Inoue ◽  
John D. Norton ◽  
Andrew D. Sharrocks
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Cellular Proliferation ◽  
Regulatory Function ◽  
Transcription Factor Family ◽  
Helix Loop Helix ◽  
Id Proteins ◽  
Proliferation And Differentiation

ABSTRACT The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.

scholarly journals Myogenic Basic Helix-Loop-Helix Proteins and Sp1 Interact as Components of a Multiprotein Transcriptional Complex Required for Activity of the Human Cardiac α-Actin Promoter

1999 ◽  
Vol 19 (4) ◽  
pp. 2577-2584 ◽  
Author(s):  
Elzbieta Biesiada ◽  
Yasuo Hamamori ◽  
Larry Kedes ◽  
Vittorio Sartorelli
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Serum Response Factor ◽  
Multiprotein Complex ◽  
Protein Protein Interactions ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Transcriptional Complex

ABSTRACT Activation of the human cardiac α-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.

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