Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation

Development ◽  
2000 ◽  
Vol 127 (8) ◽  
pp. 1593-1605 ◽  
Author(s):  
H.L. Park ◽  
C. Bai ◽  
K.A. Platt ◽  
M.P. Matise ◽  
A. Beeghly ◽  
...  
Keyword(s):  
Dna Binding ◽  
Sonic Hedgehog ◽  
Zinc Fingers ◽  
Branching Morphogenesis ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Shh Signaling ◽  
Anteroposterior Patterning ◽  
Gli Transcription Factors ◽  
Ventral Spinal Cord

The secreted factor Sonic hedgehog (SHH) is both required for and sufficient to induce multiple developmental processes, including ventralization of the CNS, branching morphogenesis of the lungs and anteroposterior patterning of the limbs. Based on analogy to the Drosophila Hh pathway, the multiple GLI transcription factors in vertebrates are likely to both transduce SHH signaling and repress Shh transcription. In order to discriminate between overlapping versus unique requirements for the three Gli genes in mice, we have produced a Gli1 mutant and analyzed the phenotypes of Gli1/Gli2 and Gli1/3 double mutants. Gli3(xt) mutants have polydactyly and dorsal CNS defects associated with ectopic Shh expression, indicating GLI3 plays a role in repressing Shh. In contrast, Gli2 mutants have five digits, but lack a floorplate, indicating that it is required to transduce SHH signaling in some tissues. Remarkably, mice homozygous for a Gli1(zfd)mutation that deletes the exons encoding the DNA-binding domain are viable and appear normal. Transgenic mice expressing a GLI1 protein lacking the zinc fingers can not induce SHH targets in the dorsal brain, indicating that the Gli1(zfd)allele contains a hypomorphic or null mutation. Interestingly, Gli1(zfd/zfd);Gli2(zfd/+), but not Gli1(zfd/zfd);Gli3(zfd/+) double mutants have a severe phenotype; most Gli1(zfd/zfd);Gli2(zfd/+) mice die soon after birth and all have multiple defects including a variable loss of ventral spinal cord cells and smaller lungs that are similar to, but less extreme than, Gli2(zfd/zfd) mutants. Gli1/Gli2 double homozygous mutants have more extreme CNS and lung defects than Gli1(zfd/zfd);Gli2(zfd/+) mutants, however, in contrast to Shh mutants, ventrolateral neurons develop in the CNS and the limbs have 5 digits with an extra postaxial nubbin. These studies demonstrate that the zinc-finger DNA-binding domain of GLI1 protein is not required for SHH signaling in mouse. Furthermore, Gli1 and Gli2, but not Gli1 and Gli3, have extensive overlapping functions that are likely downstream of SHH signaling.

Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor

1993 ◽  
Vol 13 (11) ◽  
pp. 6858-6865
Author(s):  
M W Russo ◽  
C Matheny ◽  
J Milbrandt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Zinc Fingers ◽  
Fold Increase ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domains ◽  
Cellular Factor ◽  
Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

scholarly journals Repression and Activation Domains of Rme1p Structurally Overlap, but Differ in Genetic Requirements

2002 ◽  
Vol 13 (5) ◽  
pp. 1709-1721 ◽  
Author(s):  
Anna Blumental-Perry ◽  
Weishi Li ◽  
Giora Simchen ◽  
Aaron P. Mitchell
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Structural Integrity ◽  
Zinc Finger Protein ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Functional Difference ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains

Rme1p, a repressor of meiosis in the yeast Saccharomyces cerevisiae, acts as both a transcriptional repressor and activator. Rme1p is a zinc-finger protein with no other homology to any protein of known function. The C-terminal DNA binding domain of Rme1p is essential for function. We find that mutations and progressive deletions in all three zinc fingers can be rescued by fusion ofRME1 to the DNA binding domain of another protein. Thus, structural integrity of the zinc fingers is not required for the Rme1p-mediated effects on transcription. Using a series of mutant Rme1 proteins, we have characterized domains responsible for repression and activation. We find that the minimal transcriptional repression and activation domains completely overlap and lie in an 88-amino-acid N-terminal segment (aa 61–148). An additional transcriptional effector determinant lies in the first 31 amino acids of the protein. Notwithstanding the complete overlap between repression and activation domains of Rme1p, we demonstrated a functional difference between repression and activation: Rgr1p and Sin4p are absolutely required for repression but dispensable for activation.

scholarly journals Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization

2007 ◽  
Vol 21 (6) ◽  
pp. 1297-1311 ◽  
Author(s):  
Kai Sun ◽  
Vedrana Montana ◽  
Karthikeyani Chellappa ◽  
Yann Brelivet ◽  
Dino Moras ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Receptors ◽  
Nuclear Localization ◽  
Thyroid Hormone Receptor ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Retinoid X Receptor ◽  
Common Mechanism ◽  
Dna Binding Domain ◽  
Cytoplasmic Localization

Abstract Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4α (HNF4α). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4α and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4α (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4α as well as decreased endogenous HNF4α protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor α, retinoid X receptor α, and thyroid hormone receptor β) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor α and peroxisome proliferator-activated receptor α. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

scholarly journals Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor.

1993 ◽  
Vol 13 (11) ◽  
pp. 6858-6865 ◽  
Author(s):  
M W Russo ◽  
C Matheny ◽  
J Milbrandt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Zinc Fingers ◽  
Fold Increase ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domains ◽  
Cellular Factor ◽  
Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1

1993 ◽  
Vol 13 (8) ◽  
pp. 4556-4571
Author(s):  
A L Gashler ◽  
S Swaminathan ◽  
V P Sukhatme
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Zinc Fingers ◽  
Early Response ◽  
Subcellular Fractionation ◽  
Binding Domain ◽  
Immediate Early ◽  
Regulatory Function ◽  
Dna Binding Domain ◽  
Beta Galactosidase

Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, DNA-binding, and nuclear localization activities. Deletion analysis, as well as fusions to the DNA-binding domain of GAL4, indicated that the activation potential of Egr-1 is distributed over an extensive serine/threonine-rich N-terminal domain. In addition, a novel negative regulatory function has been precisely mapped 5' of the zinc fingers: amino acids 281 to 314 are sufficient to confer the ability to repress transcription on a heterologous DNA-binding domain. Specific DNA-binding activity was shown to reside in the three zinc fingers of Egr-1, as predicted by homology to other known DNA-binding proteins. Finally, nuclear localization of Egr-1 is specified by signals in the DNA-binding domain and basic flanking sequences, as determined by subcellular fractionation and indirect immunofluorescence. Basic residues 315 to 330 confer partial nuclear localization on the bacterial protein beta-galactosidase. A bipartite signal consisting of this basic region in conjunction with either the second or third zinc finger, but not the first, suffices to target beta-galactosidase exclusively to the nucleus. Our work shows that Egr-1 is a functionally complex protein and suggests that it may play different roles in the diverse settings in which it is induced.

Two of the Five Zinc Fingers in the Zap1 Transcription Factor DNA Binding Domain Dominate Site-Specific DNA Binding†

Biochemistry ◽  
2003 ◽  
Vol 42 (4) ◽  
pp. 1053-1061 ◽  
Author(s):  
M. V. Evans-Galea ◽  
E. Blankman ◽  
D. G. Myszka ◽  
A. J. Bird ◽  
D. J. Eide ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Site Specific

scholarly journals Metal-Responsive Transcription Factor 1 (MTF-1) Activity Is Regulated by a Nonconventional Nuclear Localization Signal and a Metal-Responsive Transactivation Domain

2009 ◽  
Vol 29 (23) ◽  
pp. 6283-6293 ◽  
Author(s):  
Uschi Lindert ◽  
Mirjam Cramer ◽  
Michael Meuli ◽  
Oleg Georgiev ◽  
Walter Schaffner
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Localization Signal ◽  
Transcription Factor 1

ABSTRACT Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; “humanizing” mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

scholarly journals Mapping functional regions of transcription factor TFIIIA.

1988 ◽  
Vol 8 (4) ◽  
pp. 1684-1696 ◽  
Author(s):  
K E Vrana ◽  
M E Churchill ◽  
T D Tullius ◽  
D D Brown
Keyword(s):  
Dna Binding ◽  
Internal Control ◽  
Zinc Fingers ◽  
In Vitro Transcription ◽  
Binding Domain ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Dna Binding Domain ◽  
Protein Mass

Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.

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