The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 99-112 ◽  
Author(s):  
M. Capovilla ◽  
E.D. Eldon ◽  
V. Pirrotta
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Ectopic Expression ◽  
Regulatory Elements ◽  
In Vitro Translation ◽  
Gap Genes ◽  
Zip Family ◽  
Negative Effect ◽  
Drosophila Embryos

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Kruppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Kruppel and knirps segmentation gap genes. The analysis of the interactions between Kruppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Kruppel is in fact mediated by a negative effect of Kruppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Kruppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.

scholarly journals TFEC, a basic helix-loop-helix protein, forms heterodimers with TFE3 and inhibits TFE3-dependent transcription activation.

1993 ◽  
Vol 13 (8) ◽  
pp. 4505-4512 ◽  
Author(s):  
G Q Zhao ◽  
Q Zhao ◽  
X Zhou ◽  
M G Mattei ◽  
B de Crombrugghe
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
In Vitro Translation ◽  
Chain Gene ◽  
Adult Rats ◽  
Sequence Identity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Heavy Chain Gene Enhancer

We have identified a new basic helix-loop-helix (BHLH) DNA-binding protein, designated TFEC, which is closely related to TFE3 and TFEB. The basic domain of TFEC is identical to the basic DNA-binding domain of TFE3 and TFEB, whereas the helix-loop-helix motif of TFEC shows 88 and 85% identity with the same domains in TFE3 and TFEB, respectively. Like the other two proteins, TFEC contains a leucine zipper motif, which has a lower degree of sequence identity with homologous domains in TFE3 and TFEB than does the BHLH segment. Little sequence identity exists outside these motifs. Unlike the two other proteins, TFEC does not contain an acidic domain, which for TFE3 mediates the ability to activate transcription. Like the in vitro translation product of TFE3, the in vitro-translated TFEC binds to the mu E3 DNA sequence of the immunoglobulin heavy-chain gene enhancer. In addition, the product of cotranslation of TFEC RNA and TFE3 RNA forms a heteromeric protein-DNA complex with mu E3 DNA. In contrast to TFE3, TFEC is unable to transactivate a reporter gene linked to a promoter containing tandem copies of the immunoglobulin mu E3 enhancer motif. Cotransfection of TFEC DNA and TFE3 DNA strongly inhibits the transactivation caused by TFE3. TFEC RNA is found in many tissues of adult rats, but the relative concentrations of TFEC and TFE3 RNAs vary considerably in these different tissues. No TFEC RNA was detectable in several cell lines, including fibroblasts, myoblasts, chondrosarcoma cells, and myeloma cells, indicating that TFEC is not ubiquitously expressed.

TFEC, a basic helix-loop-helix protein, forms heterodimers with TFE3 and inhibits TFE3-dependent transcription activation

1993 ◽  
Vol 13 (8) ◽  
pp. 4505-4512
Author(s):  
G Q Zhao ◽  
Q Zhao ◽  
X Zhou ◽  
M G Mattei ◽  
B de Crombrugghe
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
In Vitro Translation ◽  
Chain Gene ◽  
Adult Rats ◽  
Sequence Identity ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Heavy Chain Gene Enhancer

We have identified a new basic helix-loop-helix (BHLH) DNA-binding protein, designated TFEC, which is closely related to TFE3 and TFEB. The basic domain of TFEC is identical to the basic DNA-binding domain of TFE3 and TFEB, whereas the helix-loop-helix motif of TFEC shows 88 and 85% identity with the same domains in TFE3 and TFEB, respectively. Like the other two proteins, TFEC contains a leucine zipper motif, which has a lower degree of sequence identity with homologous domains in TFE3 and TFEB than does the BHLH segment. Little sequence identity exists outside these motifs. Unlike the two other proteins, TFEC does not contain an acidic domain, which for TFE3 mediates the ability to activate transcription. Like the in vitro translation product of TFE3, the in vitro-translated TFEC binds to the mu E3 DNA sequence of the immunoglobulin heavy-chain gene enhancer. In addition, the product of cotranslation of TFEC RNA and TFE3 RNA forms a heteromeric protein-DNA complex with mu E3 DNA. In contrast to TFE3, TFEC is unable to transactivate a reporter gene linked to a promoter containing tandem copies of the immunoglobulin mu E3 enhancer motif. Cotransfection of TFEC DNA and TFE3 DNA strongly inhibits the transactivation caused by TFE3. TFEC RNA is found in many tissues of adult rats, but the relative concentrations of TFEC and TFE3 RNAs vary considerably in these different tissues. No TFEC RNA was detectable in several cell lines, including fibroblasts, myoblasts, chondrosarcoma cells, and myeloma cells, indicating that TFEC is not ubiquitously expressed.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

1993 ◽  
Vol 178 (1) ◽  
pp. 49-62 ◽  
Author(s):  
M J Fritzler ◽  
J C Hamel ◽  
R L Ochs ◽  
E K Chan
Keyword(s):  
Recombinant Proteins ◽  
Heavy Chain ◽  
Golgi Complex ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Cytoplasmic Dynein ◽  
In Vitro Translation ◽  
Rabbit Serum ◽  
Cell Extracts

Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4349-4358 ◽  
Author(s):  
J. Charite ◽  
W. de Graaff ◽  
D. Consten ◽  
M.J. Reijnen ◽  
J. Korving ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Positional Information ◽  
Regulatory Elements ◽  
Gene Products ◽  
Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains

1991 ◽  
Vol 11 (7) ◽  
pp. 3624-3632
Author(s):  
C Abate ◽  
D Luk ◽  
T Curran
Keyword(s):  
Transcriptional Regulation ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Regulatory Function ◽  
Activation Domain ◽  
Regulatory Domains ◽  
Heterodimeric Complex ◽  
Transcriptional Stimulation

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 4965-4976 ◽  
Author(s):  
A.J. Bendall ◽  
J. Ding ◽  
G. Hu ◽  
M.M. Shen ◽  
C. Abate-Shen
Keyword(s):  
Protein Interactions ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Homeobox Gene ◽  
Regulatory Elements ◽  
Limb Muscle ◽  
Chicken Embryos ◽  
Myogenic Activity ◽  
Myod Expression

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development - namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other's trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.

scholarly journals Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation

2003 ◽  
Vol 374 (2) ◽  
pp. 423-431 ◽  
Author(s):  
Christopher D. DEPPMANN ◽  
Tina M. THORNTON ◽  
Fransiscus E. UTAMA ◽  
Elizabeth J. TAPAROWSKY
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activator Protein 1 ◽  
Basic Leucine Zipper ◽  
Activator Protein

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

scholarly journals Specificity of Rel-inhibitor interactions in Drosophila embryos.

1995 ◽  
Vol 15 (7) ◽  
pp. 3627-3634 ◽  
Author(s):  
K Tatei ◽  
M Levine
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Function Analysis ◽  
Nuclear Localization Sequence ◽  
Sequence Motif ◽  
Composite Surface ◽  
Region I ◽  
Localization Sequence ◽  
Drosophila Embryos

The Rel family of transcription factors participate in a diverse array of processes, including acute responses to injury and infection, lymphocyte differentiation, and embryonic patterning. These proteins show homology in an extended region spanning about 300 amino acids (the Rel homology domain [RHD]). The RHD mediates both DNA binding and interactions with a family of inhibitor proteins, including I kappa B alpha and cactus. Previous studies have shown that an N-terminal region of the RHD (containing the sequence motif RXXRXRXXC) is important for DNA binding, while the C-terminal nuclear localization sequence is important for inhibitor interactions. Here we present a structure-function analysis of the Drosophila dorsal RHD. These studies identify another sequence within the RHD (region I) that is essential for inhibitor interactions. There is a tight correlation between the conservation of region I sequences and the specificity of Rel-inhibitor interactions in both flies and mammals. Point mutations in the region I sequence can uncouple DNA binding and inhibitor interactions in vitro. The phenotypes associated with the expression of a modified dorsal protein in transgenic Drosophila embryos suggest a similar uncoupling in vivo. Recent crystallographic studies suggest that the region I sequence and the nuclear localization sequence might form a composite surface which interacts with inhibitor proteins.

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