scholarly journals An amino-terminal c-myc domain required for neoplastic transformation activates transcription.

1990 ◽  
Vol 10 (11) ◽  
pp. 5914-5920 ◽  
Author(s):  
G J Kato ◽  
J Barrett ◽  
M Villa-Garcia ◽  
C V Dang
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Nuclear Dna ◽  
Neoplastic Transformation ◽  
Biochemical Properties ◽  
Regulatory Function ◽  
Ras Gene ◽  
Amino Terminal

The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.

An amino-terminal c-myc domain required for neoplastic transformation activates transcription

1990 ◽  
Vol 10 (11) ◽  
pp. 5914-5920
Author(s):  
G J Kato ◽  
J Barrett ◽  
M Villa-Garcia ◽  
C V Dang
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Nuclear Dna ◽  
Neoplastic Transformation ◽  
Biochemical Properties ◽  
Regulatory Function ◽  
Ras Gene ◽  
Amino Terminal

The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.

The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 245-255 ◽  
Author(s):  
M. Van Doren ◽  
H.M. Ellis ◽  
J.W. Posakony
Keyword(s):  
Dna Binding ◽  
Cell Fate ◽  
Protein Complexes ◽  
Competitive Inhibitor ◽  
Binding Activity ◽  
Regulatory Function ◽  
Biochemical Basis ◽  
Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

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.

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 Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

scholarly journals 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.

scholarly journals The Vitamin B 12 -Dependent Photoreceptor AerR Relieves Photosystem Gene Repression by Extending the Interaction of CrtJ with Photosystem Promoters

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Mingxu Fang ◽  
Carl E. Bauer
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Light Absorption ◽  
Growth Conditions ◽  
Binding Activity ◽  
Regulatory Function ◽  
Vitamin B ◽  
Dna Binding Activity

ABSTRACT Purple nonsulfur bacteria adapt their physiology to a wide variety of environmental conditions often through the control of transcription. One of the main transcription factors involved in controlling expression of the Rhodobacter capsulatus photosystem is CrtJ, which functions as an aerobic repressor of photosystem genes. Recently, we reported that a vitamin B 12 binding antirepressor of CrtJ called AerR is required for anaerobic expression of the photosystem. However, the mechanism whereby AerR regulates CrtJ activity is unclear. In this study, we used a combination of next-generation sequencing and biochemical methods to globally identify genes under control of CrtJ and the role of AerR in controlling this regulation. Our results indicate that CrtJ has a much larger regulon than previously known, with a surprising regulatory function under both aerobic and anaerobic photosynthetic growth conditions. A combination of in vivo chromatin immunoprecipitation-DNA sequencing (ChIP-seq) and ChIP-seq and exonuclease digestion (ChIP-exo) studies and in vitro biochemical studies demonstrate that AerR forms a 1:2 complex with CrtJ (AerR-CrtJ 2 ) and that this complex binds to many promoters under photosynthetic conditions. The results of in vitro and in vivo DNA binding studies indicate that AerR-CrtJ 2 anaerobically forms an extended interaction with the bacteriochlorophyll bchC promoter to relieve repression by CrtJ. This is contrasted by aerobic growth conditions where CrtJ alone functions as an aerobic repressor of bchC expression. These results indicate that the DNA binding activity of CrtJ is modified by interacting with AerR in a redox-regulated manner and that this interaction alters CrtJ’s function. IMPORTANCE Photoreceptors control a wide range of physiology often by regulating downstream gene expression in response to light absorption via a bound chromophore. Different photoreceptors are known to utilize a number of different compounds for light absorption, including the use of such compounds as flavins, linearized tetrapyrroles (bilins), and carotenoids. Recently, a novel class of photoreceptors that use vitamin B 12 (cobalamin) as a blue-light-absorbing chromophore have been described. In this study, we analyzed the mechanism by which the vitamin B 12 binding photoreceptor AerR controls the DNA binding activity of the photosystem regulator CrtJ. This study shows that a direct interaction between the vitamin B 12 binding photoreceptor AerR with CrtJ modulates CrtJ binding to DNA and importantly, the regulatory outcome of gene expression, as shown here with photosystem promoters.

scholarly journals Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding.

1991 ◽  
Vol 11 (10) ◽  
pp. 4918-4926 ◽  
Author(s):  
W T Pu ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Alpha Helix ◽  
Conserved Residues ◽  
Sequence Recognition ◽  
Bzip Domain ◽  
Bzip Proteins ◽  
Basic Region

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.

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 Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding

1991 ◽  
Vol 11 (10) ◽  
pp. 4918-4926
Author(s):  
W T Pu ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Alpha Helix ◽  
Conserved Residues ◽  
Sequence Recognition ◽  
Bzip Domain ◽  
Bzip Proteins ◽  
Basic Region

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.

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