Fused protein domains inhibit DNA binding by LexA.

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

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Ikaros 6 Immortalizes Murine Hematopoietic Precursors In Vitro.

Blood ◽

10.1182/blood.v106.11.4354.4354 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4354-4354

Author(s):

Anna Ruiz ◽

Hugh J.M. Brady

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Zinc Finger Protein ◽

Dominant Negative ◽

In Vitro Assays ◽

Dna Binding Domains ◽

Binding Domains ◽

Delayed Cell Death

Abstract The Ikaros transcription factor has been shown to play an important role in the differentiation of both the myeloid and lymphoid lineages. The ikaros gene encodes for a zinc finger protein containing seven exons that can be alternatively spliced generating several isoforms with differing functional properties. Isoforms with less than three DNA binding domains act as dominant negative (DN) by forming complexes with longer isoforms and interfering with their DNA binding and transcriptional activation ability. Mice heterozygous for a DN ikaros isoform develop T cell leukemia and lymphoma with 100% penetrance. Overexpression of DN Ikaros isoforms has been found in some forms of leukemias. We have previously reported overexpression of the DN Ikaros6 (Ik6) isoform in a subset of leukemia patients harboring t(4;11) translocations. In addition, we inducibly expressed Ik6 in BaF3 cells and found that Ik6 overexpression delayed cell death after IL-3 withdrawal. To further investigate the leukemogenic properties of Ik6 overexpression, we have transduced murine hematopoietic precursors with a retroviral Ik6 expression vector and have analysed the effects on proliferation and differentiation of these precursors by in vitro colony formation assays. We have found that Ik6 can immortalize murine hematopopietic precursors in these in vitro assays. We are currently analysing the leukemogenic potential of Ik6 in vivo by transplanting Ik6 expressing cell lines into NOD/SCID mice.

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Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

Journal of Virology ◽

10.1128/jvi.79.13.8661-8664.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8661-8664 ◽

Cited By ~ 8

Author(s):

Stephen Schuck ◽

Arne Stenlund

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Dna Binding Domains ◽

Binding Domains ◽

Severe Effect ◽

Origin Of Dna Replication ◽

Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

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Cooperative interactions between paired domain and homeodomain

Development ◽

10.1242/dev.122.9.2639 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2639-2650 ◽

Cited By ~ 13

Author(s):

S. Jun ◽

C. Desplan

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Target Genes ◽

Cooperative Interaction ◽

Paired Domain ◽

Cooperative Interactions ◽

Dna Binding Domains ◽

Binding Domains

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs, the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

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Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein

Physiological Genomics ◽

10.1152/physiolgenomics.00100.2009 ◽

2009 ◽

Vol 39 (3) ◽

pp. 195-201 ◽

Cited By ~ 23

Author(s):

Hee Keun Lee ◽

Mee Hyun Song ◽

Myengmo Kang ◽

Jung Tae Lee ◽

Kyoung-Ah Kong ◽

...

Keyword(s):

Dna Binding ◽

Inner Ear ◽

Molecular Mechanisms ◽

Tertiary Structure ◽

In Vitro Assays ◽

Wild Type ◽

Mutant Proteins ◽

Dna Binding Domains ◽

Binding Domains

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

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Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4970-4980.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4970-4980

Author(s):

C D Putnam ◽

C S Pikaard

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Consensus Sequence ◽

Dnase I ◽

Ribosomal Gene ◽

Minimum Requirement ◽

Dna Binding Domains ◽

Binding Domains

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.

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Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers.

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4970 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4970-4980 ◽

Cited By ~ 16

Author(s):

C D Putnam ◽

C S Pikaard

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Consensus Sequence ◽

Dnase I ◽

Ribosomal Gene ◽

Minimum Requirement ◽

Dna Binding Domains ◽

Binding Domains

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.

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Cdc13 N-Terminal Dimerization, DNA Binding, and Telomere Length Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.00515-10 ◽

2010 ◽

Vol 30 (22) ◽

pp. 5325-5334 ◽

Cited By ~ 23

Author(s):

Meghan T. Mitchell ◽

Jasmine S. Smith ◽

Mark Mason ◽

Sandy Harper ◽

David W. Speicher ◽

...

Keyword(s):

Dna Binding ◽

Telomere Length ◽

Functional Characterization ◽

Point Mutations ◽

Telomeric Dna ◽

Dna Binding Domains ◽

Binding Domains ◽

Length Regulation ◽

Telomere Length Regulation

ABSTRACT The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

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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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N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.852-860.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 852-860

Author(s):

M B Toledano ◽

D Ghosh ◽

F Trinh ◽

W J Leonard

Keyword(s):

Dna Binding ◽

Point Mutations ◽

Terminal Region ◽

Sequence Motif ◽

Structural Determinants ◽

Nf Kappa B ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Binding Specificity

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

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