scholarly journals GT-2: in vivo transcriptional activation activity and definition of novel twin DNA binding domains with reciprocal target sequence selectivity.

1996 ◽  
Vol 8 (6) ◽  
pp. 1041-1059 ◽  
Author(s):  
M Ni ◽  
K Dehesh ◽  
J M Tepperman ◽  
P H Quail
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Sequence ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Sequence Selectivity ◽  
Definition Of

Ikaros 6 Immortalizes Murine Hematopoietic Precursors In Vitro.

Blood ◽  
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.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
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.

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

2010 ◽  
Vol 30 (22) ◽  
pp. 5325-5334 ◽  
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.

Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?

1992 ◽  
Vol 103 (1) ◽  
pp. 9-14 ◽  
Author(s):  
K.A. Lee
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Experimental Evidence ◽  
Cdna Cloning ◽  
Transcriptional Activation ◽  
Effector Functions ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Elements

Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.

In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2673-2685 ◽  
Author(s):  
C. Bertuccioli ◽  
L. Fasano ◽  
S. Jun ◽  
S. Wang ◽  
G. Sheng ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Mode ◽  
Cooperative Interaction ◽  
Paired Domain ◽  
Conserved Motifs ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Segment Polarity ◽  
Functionally Impaired

The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the N- terminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.

scholarly journals Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

1993 ◽  
Vol 13 (12) ◽  
pp. 7257-7266 ◽  
Author(s):  
C Carriere ◽  
S Plaza ◽  
P Martin ◽  
B Quatannens ◽  
M Bailly ◽  
...  
Keyword(s):  
Dna Binding ◽  
Autosomal Dominant ◽  
Paired Domain ◽  
Terminal Part ◽  
Dominant Mutation ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Carboxyl Terminal

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

scholarly journals SATB1 Cleavage by Caspase 6 Disrupts PDZ Domain-Mediated Dimerization, Causing Detachment from Chromatin Early in T-Cell Apoptosis

2001 ◽  
Vol 21 (16) ◽  
pp. 5591-5604 ◽  
Author(s):  
Sanjeev Galande ◽  
Liliane A. Dickinson ◽  
I. Saira Mian ◽  
Marianna Sikorska ◽  
Terumi Kohwi-Shigematsu
Keyword(s):  
T Cell ◽  
Dna Binding ◽  
Cell Apoptosis ◽  
Pdz Domain ◽  
Dna Binding Domains ◽  
T Cell Apoptosis ◽  
Binding Domains ◽  
Caspase 6 ◽  
Loop Domains

ABSTRACT SATB1 is expressed primarily in thymocytes and orchestrates temporal and spatial expression of a large number of genes in the T-cell lineage. SATB1 binds to the bases of chromatin loop domains in vivo, recognizing a special DNA context with strong base-unpairing propensity. The majority of thymocytes are eliminated by apoptosis due to selection processes in the thymus. We investigated the fate of SATB1 during thymocyte and T-cell apoptosis. Here we show that SATB1 is specifically cleaved by a caspase 6-like protease at amino acid position 254 to produce a 65-kDa major fragment containing both a base-unpairing region (BUR)-binding domain and a homeodomain. We found that this cleavage separates the DNA-binding domains from amino acids 90 to 204, a region which we show to be a dimerization domain. The resulting SATB1 monomer loses its BUR-binding activity, despite containing both its DNA-binding domains, and rapidly dissociates from chromatin in vivo. We found this dimerization region to have sequence similarity to PDZ domains, which have been previously shown to be involved in signaling by conferring protein-protein interactions. SATB1 cleavage during Jurkat T-cell apoptosis induced by an anti-Fas antibody occurs concomitantly with the high-molecular-weight fragmentation of chromatin of ∼50-kb fragments. Our results suggest that mechanisms of nuclear degradation early in apoptotic T cells involve efficient removal of SATB1 by disrupting its dimerization and cleavage of genomic DNA into loop domains to ensure rapid and efficient disassembly of higher-order chromatin structure.

scholarly journals Fused protein domains inhibit DNA binding by LexA.

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.

scholarly journals The Activity of Mammalian brm/SNF2α Is Dependent on a High-Mobility-Group Protein I/Y-Like DNA Binding Domain

1999 ◽  
Vol 19 (6) ◽  
pp. 3931-3939 ◽  
Author(s):  
Brigitte Bourachot ◽  
Moshe Yaniv ◽  
Christian Muchardt
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
High Mobility ◽  
High Mobility Group ◽  
Binding Motif ◽  
High Mobility Group Protein ◽  
Interaction Domain ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Group Protein

ABSTRACT The mammalian SWI-SNF complex is a chromatin-remodelling machinery involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2α and BRG-1/SNF2β. These two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105Rb repression of E2F transcriptional activity. In the present study, we have examined the ability of various brm/SNF2α deletion mutants to reverse the transformed phenotype ofras-transformed fibroblasts. Deletions within the p105Rb LXCXE binding motif or the conserved bromodomain had only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105Rb interaction domain, appeared to be essential in this assay. This region was also required for cooperation of brm/SNF2α with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2α to chromatin.

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