[15] Identification of protein-protein interactions by λgt11 expression cloning

Em, Blackwood; Rn, Eisenman

doi:10.1016/0076-6879(95)54017-2

Cited by 4 publications

(3 citation statements)

References 15 publications

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“…Following electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Millipore). Denaturation and renaturation of filter-bound proteins were accomplished by immersing the membranes in 6 M guanidine HCl, gently agitating the mixture at room temperature for 10 min, and renaturing the samples by 1:1 serial dilutions with binding buffer (5,7). The membrane was washed in binding buffer for 10 min, and the filter was blocked with 3% BSA (wt/vol) for a minimum 1 h at 20°C.…”

Section: Methodsmentioning

confidence: 99%

Developmental Effects of Ectopic Expression of the Glucocorticoid Receptor DNA Binding Domain Are Alleviated by an Amino Acid Substitution That Interferes with Homeodomain Binding

Wang¹,

Préfontaine²,

Lemieux³

et al. 1999

Molecular and Cellular Biology

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Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.

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Section: Methodsmentioning

confidence: 99%

Developmental Effects of Ectopic Expression of the Glucocorticoid Receptor DNA Binding Domain Are Alleviated by an Amino Acid Substitution That Interferes with Homeodomain Binding

Wang¹,

Préfontaine²,

Lemieux³

et al. 1999

Molecular and Cellular Biology

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“…We screened a human placental cDNA expression library by a Far Western method (31) to identify proteins that interact with the carboxyl terminus of BRCA1. We found that the retinoblastoma-binding protein, RbAp46, interacts in vitro with the BRCT domain as well as with full-length BRCA1 in vivo.…”

mentioning

confidence: 99%

BRCA1 interacts with components of the histone deacetylase complex

Yarden

Brody

1999

Proc. Natl. Acad. Sci. U.S.A.

303

204

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Germ-line mutations in the BRCA1 tumorsuppressor gene are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 contains a carboxylterminal domain (BRCT) that is shared with several other proteins involved in maintaining genome integrity. In an effort to understand the function of BRCA1, we sought to isolate proteins that interact with the BRCT domain. Purified BRCT polypeptide was used as a probe to screen a human placenta cDNA expression library by Far Western analysis. Here we report that BRCA1 interacts in vivo and in vitro with the Rb-binding proteins, RbAp46 and RbAp48, as well as with Rb. Moreover, the BRCT domain associates with the histone deacetylases HDAC1 and HDAC2. These results demonstrate that BRCA1 interacts with components of the histone deacetylase complex, and therefore may explain the involvement of BRCA1 in multiple processes such as transcription, DNA repair, and recombination.More than half of families with inherited breast and ovarian cancer susceptibility are thought to harbor germ-line mutations in the BRCA1 gene. Frequent loss of the wild-type allele in tumors of mutation carriers suggests that BRCA1 acts as a tumor-suppressor gene. Surprisingly, mutations in BRCA1 in sporadic breast and ovarian cancer are extremely rare (1-3). To date, more than 600 different mutations in the BRCA1 gene have been reported (Breast Cancer Information Core: www. nhgri.nih.gov͞Intramuralresearch͞Labtransfer͞Bic͞). The majority of these are truncation mutations distributed over the entire length of the gene. Several missense mutations have also been shown to segregate with cancer susceptibility (1, 4, 5).The BRCA1 gene was isolated and mapped to human chromosome 17q21 (6). The gene encodes an 1,863-aa protein with an apparent molecular mass of 220 kDa. Only a few conserved sequence motifs have been identified in the BRCA1 protein: an amino-terminal RING finger, a carboxyl-terminal region that contains two repeats of a newly identified motif, designated BRCT (BRCA1 carboxyl terminus) domain (7), and three nuclear localization signals in the central portion of the molecule (8). However, much of the biochemical function of BRCA1 is unknown.BRCA1 is found in nuclear foci that form in a cell cycledependent manner (9, 10). Several lines of evidence suggest that BRCA1 expression is cell cycle regulated and plays a role in cell cycle checkpoints. BRCA1 mRNA is highly expressed during embryonic development and is increased in breast epithelia during pregnancy and in adult testis during the final stages of meiosis and spermatogenesis (11, 12), suggesting a role in terminal differentiation. Brca1Ϫ͞Ϫ mouse embryos die early in development from cell proliferation defects, including cell cycle arrest (13,14). In human cell lines, BRCA1 expression suppresses cell growth (15)(16)(17). The presence of the BRCT motif in BRCA1 also links it to cell cycle control. Many other cell cycle checkpoint proteins, such as the p53-binding protein (53BP1), fission yeast replication checkpoint proteins,...

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“…The dimerization of a leucine zipper was used as a proof of principle (Hu et al, 1990). The immunity assay has been applied in many publications to investigate protein-protein interactions beside the yeast two-hybrid technique (Blackwood & Eisenman, 1995;Di lallo et al, 1999a;Longo et al, 1995;Wolfe et al, 1999). Various methods to determine protein or protein-DNA interactions have been published that prevent or allow transcription Vidal & Legrain, 1999).…”

Section: Introductionmentioning

confidence: 99%