KAP-1 Corepressor Protein Interacts and Colocalizes with Heterochromatic and Euchromatic HP1 Proteins: a Potential Role for Krüppel-Associated Box–Zinc Finger Proteins in Heterochromatin-Mediated Gene Silencing

Abstract

Completed research The recurrent theme that has emerged from the structural study of proteins involved in signal transduction and transcriptional regulation is that multiple independently folded globular domains in these proteins often cooperate in macromolecular recognition. We have used the modularity of highly conserved arnino acid signature motifs as an approach to studying a protein's structure and function. The RING finger motif is a very abundant sequence motif in the protein database. This highly conserved module of approximately 50 amino acids is defined by eight cysteines and/or histidines that coordinate two zinc ions in a unique cross braced fashion. Upon cloning of BRCA1, the most recognizable signature motif in the amino acid sequence was a RING finger at the N-terminus. Moreover, genetic analysis of breast cancer kindreds identified naturally occuring germline mutations that lead to single amino acid substitutions of highly conserved residues in the RING finger and predispose individuals to disease. BAP-1, BRCA1 Associated Protein 1, is a novel, exclusively nuclear ubiquitin carboxy-terminal hydrolase that was identified by the Rauscher lab in a yeast two-hybrid interaction screen for proteins that would interact with the RING finger motif of BRCA1 (Jensen et al. Oncogene 16:1097-1112, 1998). Utilizing a putative coiled-coil structural motif located near the C-terminus of BAP-1, we performed a second, subsequent two-hybrid screen to identify additional proteins that may interact with BAP-1. Sequence from five positive clones, two independent fusions, identically matched nucleotide sequence which encodes the C-terminus of ß-catenin. Quantitative ß-gal assays of yeast extracts revealed the interaction was weak, indicating that either a larger portion of ß-catenin is necessary for optimal interaction or that the interaction might be very transient. To determine if a direct interaction occurred between BAP-1 and ß-catenin, 35 S-met labeled in vitro translates of BAP-1 and ß-catenin were tested for their ability to bind to GST fusions of ß-catenin or the C-terminus of BAP-1 in vitro, respectively, ß-catenin was observed to show some specific association with the C-terminus of BAP-1. In order to verify an in vivo interaction between BAP-1 and ß-catenin, co-immunoprecipitation experiments were employed. In order to verify an in vivo interaction between BAP-1 and ß-catenin, co-immunoprecipitation of 35 S-met labeled protein from transiently transfection of CMV driven expression plasmids containing the entire open reading frames of BAP-1 and ß-catenin into COS1 cells was employed. All attempts to co-immunoprecipitate BAP1 and ß-catenin failed under all experimental conditions (i.e. whole cell lysates vs. nuclear extracts, high salt vs. low salt, different detergents) and washing stringencies. Likewise, immunoprecipitation of endogenous BAP-1 followed by Western analysis with anti-sera against ß-catenin failed to detect in vivo interaction when performed under similar conditions. Identical David C. Schultz,...

The Bladder Tumor Suppressor Protein TERE1 (UBIAD1)Modulates Cell Cholesterol: Implications for Tumor Progression

“…We adjusted baits to ensure equivalent loading and verified similar expression levels of the FLAG-TERE1 test proteins in HEK293 cell lysates. Expression of the purified GST baits was estimated from Coomassie blue-stained gels and/or immunoblot analysis to normalize the amounts used during associations by filling with empty beads as previously described (Ryan et al, 1999). Test lysates were diluted in association buffer AB (40 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.2% NP-40, 0.5% BSA plus PIN) and precleared via 1 h incubation with empty GST beads.…”

The KRAB Domain of Zinc Finger Gene ZNF268: a Potential Transcriptional Repressor

“…As reported, repression is mediated by binding of the transcriptional corepressor KRAB-associated protein-1 (KAP-1), which is also known as KRIP-1 (KRAB-A interacting protein) or TIF1b (transcription intermediary factor 1b) (17 -19). The bound corepressor then interacts with downstream target genes, such as the protein HP1 family of nonhistone heterochromatin-associated proteins (20).…”

Keeping chromatin quiet

“…48,49 In turn, HP1 favors heterochromatin formation by bridging neighboring nucleosomes and recruiting other repressive components such as H3K9 histone methyltransferases and KAP1. 28,[50][51][52][53] An attractive idea is that remodeling by SMARCAD1 initiates replication coupled histone deacetylation and restoration of silencing by facilitating subsequent repressive modifications to lysines. Such a model can account for the observation that SMARCAD1 deficiency leads to increased histone acetylation and a decrease in H3K9 methylation and HP1 binding.…”

“…KAP1 is proposed to silence transcription by assembling HDACs and other chromatin modifiers and remodelers at specific loci. 50,51,55 Moreover, KAP1 is a component of the CAF1-HP1α complex involved in heterochromatin replication and associates with pericentric heterochromatin during the retinoic acid induced differentiation of F9 embryonal carcinoma and embryonic stem cells. 28,73 Yet loss of KAP1 does not abolish the S-phase dependent localization of SMARCAD1 to pericentric heterochromatin, suggesting that association of SMARCAD1 with heterochromatin is not critically dependent on KAP1 (Fig.…”