Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal ce-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal a-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YYl-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.The CC(AIT)6GG sequence, or CArG motif, is the core of a family of DNA regulatory elements that occur in the promoters and enhancers of genes which are subject to different regulatory controls (12,23). Included in this family of regulatory elements are the c-fos serum response element (SRE), which confers serum-inducible expression, and the skeletal a-actin muscle regulatory element (MRE), which is sufficient for muscle-specific expression when it is placed upstream from a TATA element (22,24). The serum response factor (SRF) binds to the core CArG motif of both elements, while other proteins directly bind to the sequences that flank the CArG motif (9,11,16,22,24,25). In addition, ets-related proteins bind to the c-fos SRE as part of a ternary complex with the SRF (1, 7). Complex protein-nucleic acid interactions presumably allow these elements to respond to diverse intracellular signals, but the functions of the individual factors are generally not known and contradictory findings have been reported (4, 8,10,17,20,26).Here, we report that the transcription factor YY1, also referred to as 6,14,19,21), specifically binds to the c-fos SRE and the skeletal actin MRE in vitro and that the binding of YY1 will inhibit the binding of the SRF transcription factor. YY1 overexpression represses transcription from the c-fos and skeletal actin promoters, and it appears that the repression is mediated, at least in part, by the CArG regulatory elements within these promoters. In contrast to that of YY1, SRF overexpression did not detectably alter expression from these elements; however, SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions that may result from a competition for binding to DNA. promoter (positions -296 to -323). The MRE sequence is from the chicken skeletal ...

Adult vascular smooth muscle cells dedifferentiate and reenter the cell cycle in response to growth factor stimulation. Here we describe the molecular cloning from vascular smooth muscle, the structure, and the chromosomal location of a diverged homeobox gene, Gax, whose expression is largely confined to the cardiovascular tissues of the adult. In quiescent adult rat vascular smooth muscle cells, Gax mRNA levels are down-regulated as much as 15-fold within 2 h when these cells are induced to proliferate with platelet-derived growth factor (PDGF) or serum growth factors. This reduction in Gax mRNA is transient, with levels beginning to rise between 8 and 24 h after mitogen stimulation and returning to near normal by 24 to 48 h. The Gax down-regulation is dose dependent and can be correlated with the mitogen's ability to stimulate DNA synthesis. PDGF-AA, a weak mitogen for rat vascular smooth muscle cells, did not affect Gax transcript levels, while PDGF-AB and -BB, potent mitogens for these cells, were nearly as effective as fetal bovine serum. The removal of serum from growing cells induced Gax expression fivefold within 24 h. These data suggest that Gax is likely to have a regulatory function in the G0-to-G1 transition of the cell cycle in vascular smooth muscle cells.