Co-immunoprecipitation of protein complexes.

Co-immunoprecipitation is a common method used to determine protein-protein interactions. This method typically exploits the highly specific interaction between a monoclonal antibody and a protein of interest. Providing that the antibody-protein interaction does not interfere with the ability of the protein to interact with other proteins in a cell lysate, a protein complex containing the antibody, the protein of interest, and its interacting proteins could be isolated on protein A Sepharose beads and purified from the cell lysate. This chapter details many of the techniques and associated procedures of co-immunoprecipitation and highlights many of the concerns and considerations associated with each step of this method.

Two related ARID family proteins are alternative subunits of human SWI/SNF complexes.

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.

Monoclonal antibodies reactive with the BAF155 (SMARCC1) and BAF170 (SMARCC2) components of human SWI/SNF-related complexes.

Mammalian SWI/SNF-related chromatin remodeling complexes are required for transcription controls that underlie differentiation, development, and tumor suppression. The complexes each consist of an ATPase of the SWI2/SNF2 family and approximately seven stably associated non-catalytic subunits. In spite of the importance of these complexes to biological processes, monoclonal antibodies to the various subunits have not been readily available. Mammalian complexes can vary in subunit composition, but the BAF155 (SMARCC1) subunit and a closely related protein, BAF170 (SMARCC2), appear to be ubiquitous components. Here we report the development of antibodies raised against a BAF155-derived peptide. The antibodies were raised against a single peptide of 18 amino acids. However, hybridomas expressing antibodies of two different specificities were isolated. One, designated DXD7, is specific for BAF155. The other, designated DXD12, is reactive with both BAF155 and BAF170. The antibodies are reactive against both native and denatured proteins, and are suitable for immunoprecipitation and Western blots. The DXD7 antibody is suitable additionally for immunofluorescence assays.

Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription.

The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP.

Acetylation of the adenovirus-transforming protein E1A determines nuclear localization by disrupting association with importin-alpha.

Posttranslational modifications may alter the biochemical functions of a protein by modifying associations with other macromolecules, allosterically altering intrinsic catalytic activities, or determining subcellular localization. The adenovirus-transforming protein E1A is acetylated by its cellular targets, the co-activators CREB-binding protein, p300, and p300/CREB-binding protein-associated factor in vitro and also in vivo at a single lysine residue (Lys(239)) within a multifunctional carboxyl-terminal domain necessary for both nuclear localization and interaction with the transcriptional co-repressor carboxyl-terminal binding protein (CtBP). In contrast to a previous report, we demonstrate that acetylation of Lys(239) does not disrupt CtBP binding and that 12 S E1A-mediated repression of CREB-binding protein-dependent transcription does not require recruitment of CtBP. Instead we find that the cytoplasmic fraction of E1-transformed 293 cells is enriched for acetylated E1A with relative exclusion from the nuclear compartment. Whereas wild type 12 S E1A binds importin-alpha 3, binding affinity was markedly reduced both by single amino acid substitution mutations and acetylation at Lys(239). This is the first demonstration that acetylation may alter nuclear partitioning by direct interference with nuclear import receptor recognition. The finding that the cytoplasmic fraction of E1A is acetylated indicates that E1A may exert its pleiotropic effects on cellular transformation in part by affecting cytoplasmic processes.

Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii.

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.