No abstract

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.

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function.

NIH 3T3 mouse cells were transfected with plasmids that induce efficient expression of either ('l the Rous sarcoma virus v-src gene, (ii) the chicken c-src gene, or (ii) a recombinant gene combining the 5' portion of c-src with the 3' end of v-src. Focus formation in tissue culture and formation of large colonies in soft agar did not occur in cells transfected with c-src. Cells transfected with c-src expression plasmids did not form foci but were isolated using a coselectable biological marker. They display morphological and substrate-independent growth characteristics intermediate between those of normal and v-src-transformed mouse cells, and lysates from these cells have enhanced in vitro tyrosine kinase activity. Transfection with the c-src-v-src recombinant induced focus formation with an efficiency similar to that obtained with a v-src expression plasmid. These results imply that v-src-induced transformation does not result just from overexpression of an essentially normal cellular protein but, at least in part, depends on the mutations distinguishing the cellular and viral proteins.Neoplastic transformation by pp6Ov-src appears to be correlated with its tyrosine kinase activity (for review, see ref.

Adenovirus ElA transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the celular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of ElA, which is located near the N terminus. Transformationdefective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton ElA-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by ElA is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton ElA-associated protein plays a major role in ElA-mediated cell growth control mechanisms.