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

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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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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Fused protein domains inhibit DNA binding by LexA.

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3006 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3006-3014 ◽

Cited By ~ 105

Author(s):

E A Golemis ◽

R Brent

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

In Vitro Assays ◽

Dimerization Domain ◽

Activation Domains ◽

Dna Binding Domains ◽

Binding Domains ◽

Operator Binding

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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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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Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122duringXenopusDevelopment

Journal of Biological Chemistry ◽

10.1074/jbc.m406107200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52447-52455 ◽

Cited By ~ 12

Author(s):

Garry P. Scarlett ◽

Stuart J. Elgar ◽

Peter D. Cary ◽

Anna M. Noble ◽

Robert L. Orford ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Rna Binding ◽

Transcription Activator ◽

Double Stranded Rna ◽

Binding Domains ◽

Rna Binding Domains ◽

Ccaat Box

CBTF122is a subunit of theXenopusCCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (anin vivotarget of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA bindingin vitro. In addition, these mutations alter the ability of CBTF122fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 genein vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding bothin vitroandin vivo.

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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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Fused protein domains inhibit DNA binding by LexA

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3006-3014.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3006-3014

Author(s):

E A Golemis ◽

R Brent

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

In Vitro Assays ◽

Dimerization Domain ◽

Activation Domains ◽

Dna Binding Domains ◽

Binding Domains ◽

Operator Binding

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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Interaction of C/EBPbeta and v-Myb is required for synergistic activation of the mim-1 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1316 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1316-1325 ◽

Cited By ~ 60

Author(s):

S Mink ◽

U Kerber ◽

K H Klempnauer

Keyword(s):

Transcription Factor ◽

Cell Types ◽

Factor V ◽

Binding Studies ◽

Transcription Factor Family ◽

Dna Binding Domains ◽

Binding Domains ◽

Synergistic Activation

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which activates the myelomonocyte-specific mim-1 gene, a natural myb target gene, by cooperating with members of the C/EBP transcription factor family. The finding that v-Myb, together with C/EBP, is sufficient to activate the mim-1 gene in heterologous cell types has implicated Myb and C/EBP as a bipartite molecular switch, which regulates the expression of myelomonocyte-specific genes. To understand the relationship between v-Myb and C/EBP in more detail, we have examined the molecular basis of the activation of the mim-1 promoter by v-Myb and C/EBPbeta, a member of the C/EBP transcription factor family highly expressed in myelomonocytic cells. We have identified a composite Myb and C/EBP response element which mediates synergistic activation of the mim-1 promoter by both factors and consists of closely spaced Myb- and C/EBP-binding sites. In vitro and in vivo protein-binding studies indicate that v-Myb and C/EBPbeta interact with each other via their DNA-binding domains. We show that this interaction is essential for the synergistic activation of the mim-1 promoter by v-Myb and C/EBPbeta. Our work therefore identifies C/EBPbeta as an interaction partner of v-Myb involved in myelomonocyte gene expression.

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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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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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