Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

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Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(90)90196-9 ◽

1990 ◽

Vol 1087 (2) ◽

pp. 127-136 ◽

Cited By ~ 29

Author(s):

Nili Feuerstein ◽

James J. Mond ◽

Paul R. Kinchington ◽

Robert Hickey ◽

Marja-Liisa Karjalainen Lindsberg ◽

...

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Nuclear Matrix ◽

Matrix Protein ◽

Binding Activity ◽

Nuclear Matrix Protein ◽

Dna Binding Activity ◽

A Cell

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Phosphorylation Controls Ikaros's Ability To Negatively Regulate the G1-S Transition

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2797-2807.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2797-2807 ◽

Cited By ~ 71

Author(s):

Pablo Gómez-del Arco ◽

Kazushige Maki ◽

Katia Georgopoulos

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Cell Cycle Progression ◽

Rapid Development ◽

Binding Activity ◽

Casein Kinase ◽

Cycle Progression ◽

Dna Binding Activity ◽

Increased Activity ◽

Inactivating Mutations

ABSTRACT Ikaros is a key regulator of lymphocyte proliferative responses. Inactivating mutations in Ikaros cause antigen-mediated lymphocyte hyperproliferation and the rapid development of leukemia and lymphoma. Here we show that Ikaros's ability to negatively regulate the G1-S transition can be modulated by phosphorylation of a serine/threonine-rich conserved region (p1) in exon 8. Ikaros phosphorylation in p1 is induced during the G1-S transition. Mutations that prevent phosphorylation in p1 increase Ikaros's ability to impede cell cycle progression and its affinity for DNA. Casein kinase II, whose increased activity in lymphocytes leads to transformation, is a key player in Ikaros p1 phosphorylation. We thus propose that Ikaros's activity as a regulator of the G1-S transition is controlled by phosphorylation in response to signaling events that downmodulate its DNA binding activity.

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Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1566-1577.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1566-1577

Author(s):

S K Thukral ◽

A Eisen ◽

E T Young

Keyword(s):

Transcription Factor ◽

Amino Acid ◽

Dna Binding ◽

Dimethyl Sulfate ◽

Binding Activity ◽

Single Amino Acid ◽

Minor Effect ◽

Palindromic Sequence ◽

Amino Terminal ◽

Dna Binding Activity

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

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Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4609-4617.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4609-4617

Author(s):

C K Galang ◽

C A Hauser

Keyword(s):

Dna Binding ◽

Redox Regulation ◽

Mutational Analysis ◽

Cysteine Residue ◽

Binding Activity ◽

Nf Kappa B ◽

Amino Terminal ◽

Dna Binding Activity ◽

Specific Oxidation

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

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Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1987-1995.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 1987-1995

Author(s):

A A Amin ◽

P D Sadowski

Keyword(s):

Amino Acid ◽

Dna Binding ◽

In Vitro Transcription ◽

Binding Activity ◽

Binding Domain ◽

Translation System ◽

Dna Binding Domain ◽

Amino Terminal ◽

Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

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Imatinib Induced Re-Activation of FoxO3 Transcription Factor in CML Is Responsible for the Induction of a Quiescent Status of CD34 Leukaemic Progenitor Cells.

Blood ◽

10.1182/blood.v112.11.1090.1090 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1090-1090

Author(s):

Daniela Cilloni ◽

Cristina Panuzzo ◽

Francesca Messa ◽

Francesca Arruga ◽

Enrico Bracco ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Dna Binding ◽

Progenitor Cells ◽

Binding Activity ◽

Mrna Levels ◽

Dna Binding Activity ◽

Cd34 Cells

Abstract The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. In CML cells the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO3 in Bcr-Abl induced apoptotic arrest and cell growth and the effect of imatinib (IM) induced re-activation of FoxO3 activity in CML progenitor cells. BM cells were collected from 52 CML patients and 20 healthy donors. The expression level of FoxO3 was tested by RQ-PCR. The protein amount and localization was analyzed by Western blot and immunofluorescence, DNA binding activity was measured by EMSA. In addition, FoxO3 was analyzed in CML primary cells and CD34+ cells after IM incubation. Cell cycle and the expression levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization, was measured by FACS in CD34+ cell population. In addition K562 cells was transfected with pECE-FoxO3 to clarify FoxO3 effects on cell growth and apoptosis. Finally we used our already set up model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl to study the pathway leading to FoxO3 inactivation. We found that, despite either FoxO3 mRNA levels or protein amount are similar in CML cells compared to controls, FoxO3 protein is equally distributed in the nucleus and cytoplasm in controls but it is completely cytoplasmatic in CML cells and it enters the nucleus during in vivo IM treatment or in vitro IM incubation. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears after IM treatment. Moreover FoxO3 overexpression in transfected cells results into a 49±9 % reduction of proliferation which was further reduced of 75±5 % after IM incubation. Furthermore, we demonstrated that IM incubation results into the reactivation of FoxO3 in Ph+ CD34+ cells inducing quiescence into this population as demonstrated by the comparison of cell cycle kinetics and by a decreased expression of CD47. Finally, the progeny obtained from the crossbreeding of Bcr-Abl flies and flies transgenic for FoxO showed a rescue of FoxO phenotype demonstrating that FoxO inactivation is Bcr-Abl mediated. Overall, these in vitro and in vivo experiments suggest that FoxO3 is inactivated in CML cells and its delocalization is mainly dependant from Bcr-Abl activity. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. On the other side, FoxO3 re-activation induced by IM results into a quiescence of Bcr-Abl CD34+ progenitor cells, which raises a hypothesis that FoxO3 could play a role in IM resistance. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

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The DNA Binding Activity of C/EBP Transcription Factors Is Regulated in the G Phase of the Hepatocyte Cell Cycle

Journal of Biological Chemistry ◽

10.1074/jbc.270.30.18123 ◽

1995 ◽

Vol 270 (30) ◽

pp. 18123-18132 ◽

Cited By ~ 111

Author(s):

Basabi Rana ◽

Yuhong Xie ◽

David Mischoulon ◽

Nancy L. R. Bucher ◽

Stephen R. Farmer

Keyword(s):

Cell Cycle ◽

Transcription Factors ◽

Dna Binding ◽

Binding Activity ◽

Dna Binding Activity

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M-phase-specific phosphorylation of the POU transcription factor GHF-1 by a cell cycle-regulated protein kinase inhibits DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6694 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6694-6701 ◽

Cited By ~ 44

Author(s):

C Caelles ◽

H Hennemann ◽

M Karin

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Protein Kinase ◽

Dna Binding ◽

Binding Activity ◽

Dependent Manner ◽

Mitotic Kinase ◽

Dna Binding Activity ◽

M Phase ◽

A Cell

GHF-1 is a member of the POU family of homeodomain proteins. It is a cell-type-specific transcription factor responsible for determination and expansion of growth hormone (GH)- and prolactin-expressing cells in the anterior pituitary. It was previously suggested that cyclic AMP (cAMP)-responsive protein kinase A (PKA) phosphorylates GHF-1 at a site within the N-terminal arm of its homeodomain, thereby inhibiting its binding to the GH promoter. These results, however, are inconsistent with the physiological stimulation of GH production by the cAMP pathway. As reported here, cAMP agonists and PKA do not inhibit GHF-1 activity in living cells and although they stimulate the phosphorylation of GHF-1, the inhibitory phosphoacceptor site within the homeodomain is not affected. Instead, this site, Thr-220, is subject to M-phase-specific phosphorylation. As a result, GHF-1 DNA binding activity is transiently inhibited during the M phase. This activity is regained once cells enter G1, a phase during which GHF-1 phosphorylation is minimal. Thr-220 of GHF-1 is the homolog of the mitotic phosphoacceptor site responsible for the M-phase-specific inhibition of Oct-1 DNA binding Ser-382. As this site is conserved in all POU proteins, it appears that all members of this group are similarly regulated. A specific kinase activity distinct in its substrate specificity and susceptibility to inhibitors from the Cdc2 mitotic kinase or PKA was identified in extracts of mitotic cells. This novel activity could be involved in regulating the DNA binding activity of all POU proteins in a cell cycle-dependent manner.

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Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3129 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3129-3137 ◽

Cited By ~ 53

Author(s):

M Maher ◽

F Cong ◽

D Kindelberger ◽

K Nasmyth ◽

S Dalton

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Cell Cycle Regulation ◽

Ternary Complex ◽

Binding Activity ◽

Transcriptional Level ◽

Dna Binding Activity ◽

Cycle Regulation ◽

Ternary Complex Factor

Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae. We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation. Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutation which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo. A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex. A point mutation in the CLB2 UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control of CLB2 transcription. We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.

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