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

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.

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

Development ◽

10.1242/dev.122.9.2673 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2673-2685 ◽

Cited By ~ 8

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7257 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7257-7266 ◽

Cited By ~ 64

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.

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Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro

AIDS ◽

10.1097/00002030-200202150-00003 ◽

2002 ◽

Vol 16 (3) ◽

pp. 329-339 ◽

Cited By ~ 17

Author(s):

Luzia Maria de Oliveira Pinto ◽

Hervé Lecoeur ◽

Eric Ledru ◽

Christophe Rapp ◽

Olivier Patey ◽

...

Keyword(s):

T Cell ◽

Cell Apoptosis ◽

Therapy Regimen ◽

T Cell Apoptosis ◽

Lack Of Control

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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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Cytokine-dependent regulation of NADPH oxidase activity and the consequences for activated T cell homeostasis

Journal of Experimental Medicine ◽

10.1084/jem.20082851 ◽

2009 ◽

Vol 206 (7) ◽

pp. 1515-1523 ◽

Cited By ~ 33

Author(s):

Divya Purushothaman ◽

Apurva Sarin

Keyword(s):

T Cells ◽

T Cell ◽

Nadph Oxidase ◽

Cell Apoptosis ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Activated T Cells ◽

T Cell Apoptosis ◽

Mitochondrial Superoxide ◽

Activated T Cell

Cellular dependence on growth factors for survival is developmentally programmed and continues in adult metazoans. Antigen-activated T cell apoptosis in the waning phase of the immune response is thought to be triggered by depletion of cytokines from the microenvironment. T cell apoptosis resulting from cytokine deprivation is mediated by reactive oxygen species (ROS), but their source and position in the apoptotic cascade is poorly understood. RNA interference approaches implicated the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neglect-induced apoptosis in T cells. Using mice deficient for the catalytic subunit gp91phox to characterize the molecular link to activated T cell apoptosis, we show that gp91phox-deficient T (T−/−) cells generated mitochondrial superoxide but had diminished hydrogen peroxide production in response to neglect, which, in turn, regulated Jun N-terminal kinase–dependent Bax activation and apoptosis. Activated T−/− cells were distinguished by improved survival after activation by superantigens in vivo, adoptive transfers into congenic hosts, and higher recall responses after immunization. Thus, the NADPH oxidase may regulate adaptive immunity in addition to its previously well-characterized role in the innate response.

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Progression of intracranial glioma disrupts thymic homeostasis and induces T-cell apoptosis in vivo

Cancer Immunology Immunotherapy ◽

10.1007/s00262-008-0508-3 ◽

2008 ◽

Vol 57 (12) ◽

pp. 1807-1816 ◽

Cited By ~ 12

Author(s):

Abdeljabar El Andaloussi ◽

Yu Han ◽

Maciej S. Lesniak

Keyword(s):

T Cell ◽

Cell Apoptosis ◽

T Cell Apoptosis

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Locally produced C5a binds to T cell–expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis

Blood ◽

10.1182/blood-2008-04-151068 ◽

2008 ◽

Vol 112 (5) ◽

pp. 1759-1766 ◽

Cited By ~ 163

Author(s):

Peter N. Lalli ◽

Michael G. Strainic ◽

Min Yang ◽

Feng Lin ◽

M. Edward Medof ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Apoptosis ◽

Cell Expansion ◽

Immune Cell ◽

Regulatory Protein ◽

T Cell Apoptosis ◽

T Cell Expansion

Abstract Our recent studies have shown that immune cell–produced complement provides costimulatory and survival signals to naive CD4+ T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this, we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein, decay accelerating factor (DAF), and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis, whereas T-cell expansion induced by C3−/− APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a, which through binding to T cell–expressed C5aR, enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death.

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