An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos.

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.

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

Development ◽

10.1242/dev.124.17.3263 ◽

1997 ◽

Vol 124 (17) ◽

pp. 3263-3272 ◽

Cited By ~ 11

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.

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Evolution of the activation domain in a Hox transcription factor

The International Journal of Developmental Biology ◽

10.1387/ijdb.180151sb ◽

2018 ◽

Vol 62 (11-12) ◽

pp. 745-753 ◽

Cited By ~ 2

Author(s):

Ying Liu ◽

Annie Huang ◽

Rebecca M. Booth ◽

Gabriela Geraldo Mendes ◽

Zabeena Merchant ◽

...

Keyword(s):

Transcription Factor ◽

Intrinsically Disordered Protein ◽

Transcription Activation ◽

Specific Protein ◽

Amino Acid Sequences ◽

Activation Domain ◽

Mutant Proteins ◽

Activation Domains ◽

Intrinsically Disordered

Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved.

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The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis

Development ◽

10.1242/dev.126.24.5771 ◽

1999 ◽

Vol 126 (24) ◽

pp. 5771-5783 ◽

Cited By ~ 19

Author(s):

S.E. Quaggin ◽

L. Schwartz ◽

S. Cui ◽

P. Igarashi ◽

J. Deimling ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Cell Types ◽

Perinatal Period ◽

Branching Morphogenesis ◽

Bhlh Transcription Factor ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Kidney Morphogenesis

Epithelial-mesenchymal interactions are required for the development of all solid organs but few molecular mechanisms that underlie these interactions have been identified. Pod1 is a basic-helix-loop-helix (bHLH) transcription factor that is highly expressed in the mesenchyme of developing organs that include the lung, kidney, gut and heart and in glomerular visceral epithelial cells (podocytes). To determine the function of Pod1 in vivo, we have generated a lacZ-expressing null Pod1 allele. Null mutant mice are born but die in the perinatal period with severely hypoplastic lungs and kidneys that lack alveoli and mature glomeruli. Although Pod1 is exclusively expressed in the mesenchyme and podocytes, major defects are observed in the adjacent epithelia and include abnormalities in epithelial differentiation and branching morphogenesis. Pod1 therefore appears to be essential for regulating properties of the mesenchyme that are critically important for lung and kidney morphogenesis. Defects specific to later specialized cell types where Pod1 is expressed, such as the podocytes, were also observed, suggesting that this transcription factor may play multiple roles in kidney morphogenesis.

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Dissecting the temporal requirements for homeotic gene function

Development ◽

10.1242/dev.120.7.1983 ◽

1994 ◽

Vol 120 (7) ◽

pp. 1983-1995 ◽

Cited By ~ 16

Author(s):

J. Castelli-Gair ◽

S. Greig ◽

G. Micklem ◽

M. Akam

Keyword(s):

Gene Expression ◽

Nervous System ◽

Gene Function ◽

Cell Types ◽

Homeotic Gene ◽

Homeotic Genes ◽

Bithorax Complex ◽

Cell Type ◽

Homeotic Gene Expression

Homeotic genes confer identity to the different segments of Drosophila. These genes are expressed in many cell types over long periods of time. To determine when the homeotic genes are required for specific developmental events we have expressed the Ultrabithorax, abdominal-A and Abdominal-Bm proteins at different times during development using the GAL4 targeting technique. We find that early transient homeotic gene expression has no lasting effects on the differentiation of the larval epidermis, but it switches the fate of other cell types irreversibly (e.g. the spiracle primordia). We describe one cell type in the peripheral nervous system that makes sequential, independent responses to homeotic gene expression. We also provide evidence that supports the hypothesis of in vivo competition between the bithorax complex proteins for the regulation of their down-stream targets.

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Exportin Crm1 is important for Swi6 nucleocytoplasmic shuttling and MBF transcription activation in Saccharomyces cerevisiae

10.1101/2020.06.17.155598 ◽

2020 ◽

Author(s):

Kenneth D. Belanger ◽

William T. Yewdell ◽

Matthew F. Barber ◽

Amy N. Russo ◽

Mark A. Pettit ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Transcription Factor ◽

Nuclear Export ◽

Intracellular Localization ◽

Nuclear Export Signal ◽

Transcription Activation ◽

Cell Phenotype ◽

Export Signal

AbstractThe Swi6 protein acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. While the process of Swi6 nuclear import is well understood, mechanisms underlying its nuclear export remain unclear. Here we investigate Swi6 nuclear export and its impact on Swi6 function. We show that the exportin Crm1, in addition to three other karyopherins previously shown to affect Swi6 localization, is important for Swi6 nuclear export and activity. A truncation of Swi6 that removes a putative Crm1 nuclear export signal results in the loss of changes in nucleocytoplasmic Swi6 localization that normally occur during progression through the cell cycle. Mutagenesis of the NES-like sequence or removal of Crm1 activity using leptomycin B results in a similar decrease in nuclear export as cells enter S-phase. Using two-hybrid analysis, we also show that Swi6 associates with Crm1 in vivo. Alteration of the Crm1 NES in Swi6 results in a decrease in MBF-mediated gene expression, but does not affect expression of an SBF reporter, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the Crm1 NES in Swi6 results in cells that are larger than wild type, but not to the extent of those with a complete Swi6 deletion. Expressing a Swi6 NES mutant in combination with a deletion of Msn5, an exportin involved in Swi6 nuclear export and specifically affecting SBF activation, further increases the large cell phenotype, but still not to the extent observed in a Swi6 deletion mutant. These data suggest that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal and influences expression of a different subset of Swi6-controlled genes.Summary StatementPrecise intracellular localization is important for the proper activity of proteins. Here we provide evidence that the Swi6 transcription factor important for cell cycle progression shuttles between the cell nucleus and cytoplasm, its nuclear export is important for its activity, and that it contains a nuclear export signal (NES) recognized by the Crm1 nuclear transport factor.

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Genetics ◽

10.1093/genetics/153.4.1573 ◽

1999 ◽

Vol 153 (4) ◽

pp. 1573-1581 ◽

Cited By ~ 2

Author(s):

Susanna Chou ◽

Sukalyan Chatterjee ◽

Mark Lee ◽

Kevin Struhl

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Large Subunit ◽

Yeast Cells ◽

Specific Cell ◽

Wild Type ◽

Activation Domains ◽

Cycle Arrest ◽

General Transcription Factor

Abstract The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

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Dimerization through the helix-loop-helix motif enhances phosphorylation of the transcription activation domains of myogenin

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6232-6243.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6232-6243

Author(s):

J Zhou ◽

E N Olson

Keyword(s):

Transcriptional Activity ◽

Transcription Activation ◽

Bhlh Protein ◽

Specific Gene ◽

Phosphorylation Sites ◽

Gene Products ◽

Activation Domains ◽

Helix Loop Helix ◽

Carboxyl Terminal ◽

Bhlh Proteins

The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization-dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability of E2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcriptional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression.

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Mitochondrial Calcium Uptake Capacity Modulates Neocortical Excitability

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2013.61 ◽

2013 ◽

Vol 33 (7) ◽

pp. 1115-1126 ◽

Cited By ~ 23

Author(s):

Basavaraju G Sanganahalli ◽

Peter Herman ◽

Fahmeed Hyder ◽

Sridhar S Kannurpatti

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Cell Types ◽

Dependent Manner ◽

Evoked Responses ◽

Blood Oxygen Level ◽

Calcium Uniporter ◽

Sensory Evoked

Local calcium (Ca2 +) changes regulate central nervous system metabolism and communication integrated by subcellular processes including mitochondrial Ca2 + uptake. Mitochondria take up Ca2 + through the calcium uniporter (mCU) aided by cytoplasmic microdomains of high Ca2 +. Known only in vitro, the in vivo impact of mCU activity may reveal Ca2 + -mediated roles of mitochondria in brain signaling and metabolism. From in vitro studies of mitochondrial Ca2 + sequestration and cycling in various cell types of the central nervous system, we evaluated ranges of spontaneous and activity-induced Ca2 + distributions in multiple subcellular compartments in vivo. We hypothesized that inhibiting (or enhancing) mCU activity would attenuate (or augment) cortical neuronal activity as well as activity-induced hemodynamic responses in an overall cytoplasmic and mitochondrial Ca2 + -dependent manner. Spontaneous and sensory-evoked cortical activities were measured by extracellular electrophysiology complemented with dynamic mapping of blood oxygen level dependence and cerebral blood flow. Calcium uniporter activity was inhibited and enhanced pharmacologically, and its impact on the multimodal measures were analyzed in an integrated manner. Ru360, an mCU inhibitor, reduced all stimulus-evoked responses, whereas Kaempferol, an mCU enhancer, augmented all evoked responses. Collectively, the results confirm aforementioned hypotheses and support the Ca2 + uptake-mediated integrative role of in vivo mitochondria on neocortical activity.

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