Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

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Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6056-6067.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6056-6067

Author(s):

M Tanaka ◽

W Herr

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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

Biochemical Journal ◽

10.1042/bj20030455 ◽

2003 ◽

Vol 374 (2) ◽

pp. 423-431 ◽

Cited By ~ 26

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.

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Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715010287 ◽

2015 ◽

Vol 71 (8) ◽

pp. 1768-1776 ◽

Cited By ~ 8

Author(s):

Xiaojiao Fan ◽

Xu Zhang ◽

Yuwei Zhu ◽

Liwen Niu ◽

Maikun Teng ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Response Regulator ◽

Regulatory System ◽

Binding Domain ◽

Dna Binding Domain ◽

Promoter Regions ◽

Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

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Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding

Journal of Virology ◽

10.1128/jvi.00772-08 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7298-7305 ◽

Cited By ~ 21

Author(s):

Juan Cardenas-Mora ◽

Jonathan E. Spindler ◽

Moon Kyoo Jang ◽

Alison A. McBride

Keyword(s):

Dna Binding ◽

Fluorescent Protein ◽

Binding Domain ◽

Bovine Papillomavirus ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Mitotic Chromosomes ◽

Dimerization Domain ◽

E2 Protein ◽

Binding Function

ABSTRACT The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutated in this domain do not interact with Brd4. However, we found that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminated the DNA binding function of E2 had no effect on the ability of E2 to interact with Brd4, but an E2 protein with a mutation that disrupted C-terminal dimerization bound Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domains were replaced with the dimeric DNA binding domain of EBNA-1 or Gal4 bound efficiently to the Brd4 protein, but the replacement of the E2 C-terminal domain with a monomeric red fluorescent protein did not rescue efficient Brd4 binding. Thus, E2 bound to Brd4 most efficiently as a dimer. To prove this finding further, the E2 DNA binding domain was replaced with an FKBP12-derived domain in which dimerization was regulated by a bivalent ligand. This fusion protein bound Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 was required. Correspondingly, E2 proteins that could dimerize were able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.

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The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4791 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4791-4802 ◽

Cited By ~ 132

Author(s):

H P Harding ◽

M A Lazar

Keyword(s):

Retinoic Acid ◽

Dna Binding ◽

Binding Site ◽

Binding Domain ◽

Luciferase Reporter ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Direct Repeats ◽

Basal Transcription ◽

C Terminus

Rev-Erb is an orphan nuclear receptor which binds as a monomer to the thyroid/retinoic acid receptor half-site AGGTCA flanked 5' by an A/T-rich sequence, referred to here as a Rev monomer site. Fusion of Rev-Erb to the DNA binding domain of yeast GAL4 strongly repressed basal transcription of a GAL4-luciferase reporter gene as a result of the presence of a C-terminal domain containing both the hinge and heptad repeat regions. Nevertheless, wild-type Rev-Erb did not repress basal transcription from the Rev monomer binding site. Therefore, a DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb may function on a different site as a dimer. This approach identified sequences containing two Rev monomer sites arranged as direct repeats with the AGGTCA motifs separated by 2 bp (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. The DNA binding domain contained all of the determinants for Rev-DR2-specific homodimerization. Rev-Erb bound cooperatively as a homodimer to Rev-DR2, and this interaction was 5 to 10 times more stable than Rev-Erb monomer binding to the Rev monomer site. Functionally, Rev-Erb markedly repressed the basal activity of a variety of promoters with a strong Rev-DR2 specificity. The C terminus was required for this repression, consistent with the GAL4 results. However, the Rev-DR2 specificity did not require the C terminus in vivo, since fusion of C-terminally truncated Rev-Erb to a heterologous transactivation domain created a transcriptional activator specific for Rev-DR2. In addition to idealized Rev-DR2 sites, Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily members by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element.

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Functional Dissection of Human and Mouse POT1 Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.01352-08 ◽

2008 ◽

Vol 29 (2) ◽

pp. 471-482 ◽

Cited By ~ 69

Author(s):

Wilhelm Palm ◽

Dirk Hockemeyer ◽

Tatsuya Kibe ◽

Titia de Lange

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Dna Damage Response ◽

Binding Domain ◽

Dna Binding Domain ◽

Telomeric Dna ◽

C Terminus ◽

Damage Response ◽

End Resection

ABSTRACT The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5′-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5′-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5′-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.

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Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

Journal of Bacteriology ◽

10.1128/jb.00318-06 ◽

2006 ◽

Vol 188 (14) ◽

pp. 5167-5176 ◽

Cited By ~ 18

Author(s):

Deborah R. Yoder-Himes ◽

Lee Kroos

Keyword(s):

Dna Binding ◽

Response Regulator ◽

Myxococcus Xanthus ◽

Cell Movement ◽

Binding Domain ◽

Dna Binding Domain ◽

Consensus Binding Site ◽

Multicellular Development

ABSTRACT The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Ω4400. To determine if FruA is a direct regulator of Ω4400 transcription, a combination of in vivo and in vitro experiments was performed. Ω4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter −35 region in vitro. Mutations between bp −86 and −77 greatly reduced binding. One of these mutations had been shown previously to reduce Ω4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Ω4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Ω4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp −80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp −86 and −77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain.

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Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1002906 ◽

2012 ◽

Vol 8 (9) ◽

pp. e1002906 ◽

Cited By ~ 18

Author(s):

Clinton R. Paden ◽

J. Craig Forrest ◽

Scott A. Tibbetts ◽

Samuel H. Speck

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain

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Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

Journal of Bacteriology ◽

10.1128/jb.00530-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 4984-4993 ◽

Cited By ~ 23

Author(s):

Jason R. Wickstrum ◽

Jeff M. Skredenske ◽

Ana Kolin ◽

Ding J. Jin ◽

Jianwen Fang ◽

...

Keyword(s):

Dna Binding ◽

Transcription Activation ◽

In Vitro Transcription ◽

Binding Domain ◽

Receptor Protein ◽

Dna Binding Domain ◽

Dimerization Domain ◽

Half Site

ABSTRACT The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

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GATA-1 associates with and inhibits p53

Blood ◽

10.1182/blood-2008-10-180489 ◽

2009 ◽

Vol 114 (1) ◽

pp. 165-173 ◽

Cited By ~ 24

Author(s):

Cecelia D. Trainor ◽

Caroline Mas ◽

Patrick Archambault ◽

Paola Di Lello ◽

James G. Omichinski

Keyword(s):

Dna Binding ◽

Mutational Analysis ◽

Binding Domain ◽

Erythroid Cell ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Nuclear Condensation ◽

P53 Pathway ◽

In Vitro Interaction

Abstract In addition to orchestrating the expression of all erythroid-specific genes, GATA-1 controls the growth, differentiation, and survival of the erythroid lineage through the regulation of genes that manipulate the cell cycle and apoptosis. The stages of mammalian erythropoiesis include global gene inactivation, nuclear condensation, and enucleation to yield circulating erythrocytes, and some of the genes whose expression are altered by GATA-1 during this process are members of the p53 pathway. In this study, we demonstrate a specific in vitro interaction between the transactivation domain of p53 (p53TAD) and a segment of the GATA-1 DNA-binding domain that includes the carboxyl-terminal zinc-finger domain. We also show by immunoprecipitation that the native GATA-1 and p53 interact in erythroid cells and that activation of p53-responsive promoters in an erythroid cell line can be inhibited by the overexpression of GATA-1. Mutational analysis reveals that GATA-1 inhibition of p53 minimally requires the segment of the GATA-1 DNA-binding domain that interacts with p53TAD. This inhibition is reciprocal, as the activation of a GATA-1–responsive promoter can be inhibited by p53. Based on these findings, we conclude that inhibition of the p53 pathway by GATA-1 may be essential for erythroid cell development and survival.

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