Mechanism of subunit interaction and DNA binding of the heterotrimeric CCAAT binding factor CBF

Many eukaryotic promoters contain a CCAAT element at a site close ($-$80 to $-$120) to the transcription initiation site. CBF (CCAAT Binding Factor), also called NF-Y and CP1, was initially identified as a transcription factor binding to such sites in the promoters of the Type I collagen, albumin and MHC class II genes. CBF is a heteromeric transcription factor and purification and cloning of two of the subunits, CBF-A and CBF-B revealed that it was evolutionarily conserved with striking sequence identities with the yeast polypeptides HAP3 and HAP2, which are components of a CCAAT binding factor in yeast. Recombinant CBF-A and CBF-B however failed to bind to DNA containing CCAAT sequences. Biochemical experiments led to the identification of a third subunit, CBF-C which co-purified with CBF-A and complemented the DNA binding of recombinant CBF-A and CBF-B. We have recently isolated CBF-C cDNAs and have shown that bacterially expressed purified CBF-C binds to CCAAT containing DNA in the presence of recombinant CBF-A and CBF-B. Our experiments also show that a single molecule each of all the three subunits are present in the protein-DNA complex. Interestingly, CBF-C is also evolutionarily conserved and the conserved domain between CBF-C and its yeast homolog HAP5 is sufficient for CBF-C activity. Using GST-pulldown experiments we have demonstrated the existence of protein-protein interaction between CBF-A and CBF-C in the absence of CBF-B and DNA. CBF-B on other hand, requires both CBF-A and CBF-C to form a ternary complex which then binds to DNA. Mutational studies of CBF-A have revealed different domains of the protein which are involved in CBF-C interaction and CBF-B interaction. In addition, CBF-A harbors a domain which is involved in DNA recognition along with CBF-B. Dominant negative analogs of CBF-A have also substantiated our initial observation of assembly of CBF subunits. Our studies define a novel DNA binding structure of heterotrimeric CBF, where the three subunits of CBF follow a particular pathway of assembly of subunits that leads to CBF binding to DNA and activating transcription. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

Molecular interactions of the central conserved domain of p53

p53 mutations are the most commonly observed genetic alterations in human cancers to date. A majority of these point mutations cluster in four evolutionarily conserved domains spanning amino acids 100-300. This region of p53 has been called its central conserved, or conformational domain. This domain of p53 is also targeted by the SV40 T antigen. Mutation, as well as interaction with SV40 T antigen results in inactivation of p53. We hypothesized that mutations and SV40 T antigen disrupt p53 function by interfering with the molecular interactions of the central conserved domain. Using a chimeric protein consisting of the central conserved domain of wild-type p53 (amino acids 115-295) and a protein A affinity tail, we isolated several cellular proteins that interact specifically with this domain of p53. These proteins range in size from 30K to 90K M$\rm\sb{r}.$ We also employed the p53 fusion protein to demonstrate that the central conserved domain of p53 possesses sequence-specific DNA-binding activity. Interestingly, the cellular proteins binding to the central conserved domain of p53 enhance the sequence-specific DNA-binding activity of full length p53. Partial purification of the individual proteins binding to the conformational domain of p53 by utilizing a sodium chloride step-gradient enabled further characterization of two proteins: (1) a 42K M$\rm\sb{r}$ protein that eluted at 0.5M NaCl, and bound DNA nonspecifically, and (2) a 35K M$\rm\sb{r}$ protein eluting into the 1.0M NaCl fraction, capable of enhancing the sequence-specific DNA-binding activity of p53. In order to determine the physiologic relevance of the molecular interactions of the conformational domain of p53, we examined the biochemical processes underlying the TNF-$\alpha$ mediated growth suppression of the NSCLC cell line H460. While growth suppression was accompanied by enhanced sequence-specific p53-DNA binding activity in TNF-$\alpha$ treated H460 nuclei, there was no increase in p53 protein levels. Furthermore, p35 was upregulated in TNF-$\alpha$ treated H460 cells, suggesting that the enhanced p53-DNA binding seen in these cells may be mediated by p35. Our studies define two novel interactions involving the central conserved domain of p53 that appear to be functionally relevant: (1) sequence-specific DNA-binding, and (2) interaction with other cellular proteins. ^

The identification and regulation of a novel interaction occurring between $\beta$-catenin and the actin -bundling protein fascin

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesion, catenins have more recently been indicated to participate in cell and developmental signaling pathways. $\beta$-catenin, for example, associates directly with receptor tyrosine kinases and transcription factors such as LEF-1/TCF, and tranduces developmental signals within the Wnt pathway. $\beta$-catenin also appear to a role in regulating cell proliferation via its interaction with the tumor supressor protein APC. I have employed the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to $\beta$-catenin's central Armadillo-repeat domain. The $\beta$-catenin-fascin interaction exists in cell lines as well as in animal brain tissues as revealed by immunoprecipitation analysis, and substantiated in vitro with purified proteins. Fascin additionally binds to plakoglobin, which contains a more divergent Armadillo-repeat domain. Fascin and E-cadherin utilize a similar binding-site within $\beta$-catenin, such that they form mutually exclusive complexes with $\beta$-catenin. Fascin and $\beta$-catenin co-localize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. Total immunoprecipitable b-catein has several isoforms, only the hyperphosphorylated isoform 1 associated with fascin. An increased $\beta$-catenin-fascin interaction was observed in HGF stimulated cells, and in Xenopus embryos injected with src kinase RNAs. The increased $\beta$-catenin association with fascin is correlated with increased levels of $\beta$-catenin phosphorylation. $\beta$-catenin, but not fascin, can be readily phosphorylated on tyrosine in vivo following src injection of embryos, or in vitro following v-src addition to purified protein components. These observations suggest a role of $\beta$-catenin phosphorylation in regulating its interaction with fascin, and src kinase may be an important regulator of the $\beta$-catenin-fascin association in vivo. The $\beta$-catenin-fascin interaction represents a novel catenin complex, that may conceivably regulate actin cytoskeletal structures, cell adhesion, and cellular motility, perhaps in a coordinate manner with its functions in cadherin and APC complexes. ^