The transcription factor B-Myb is a cell-cycle regulated phosphoprotein involved in cell cycle progression through the transcriptional regulation of many genes. In this study, we show that the ...promoter of the fibroblast growth factor-4 (FGF-4) gene is strongly activated by B-Myb in HeLa cells and it can serve as a novel diagnostic tool for assessing B-Myb activity. Specifically, B-Myb deletion mutants were examined and domains of B-Myb required for activation of the FGF-4 promoter were identified. Using phosphorylation-deficient mutant forms of B-Myb, we also show that phosphorylation is essential for B-Myb activity. Moreover, a mutant form of B-Myb, which lacks all identified phosphorylation sites and which has little activity, can function as a dominant-negative and suppress wild-type B-Myb activity. Acetylation is another post-translational modification known to affect the activity of other Myb family members. We show that B-Myb is acetylated by the co-activator p300. We also show that the bromo and histone acetyltransferase domains of p300 are sufficient to interact with and acetylate B-Myb. These data indicate that phosphorylation of B-Myb is an essential modification for activity and that acetylation of B-Myb may play a role in B-Myb activity.
The expression of transforming growth factor-beta2 (TGF-beta2) appears to play a strong role in the establishment and progression of glial tumors. In particular, elevated expression of TGF-beta2 ...appears to be responsible for the impaired cell-mediated immunity often observed in patients with a glioblastoma. This study examined the regulation of the TGF-beta2 at the transcriptional level in the U87MG glioblastoma cell line. We demonstrate that a cAMP response element/activating transcription factor (CRE/ATF) site and an E-box motif located just upstream of the transcription start site are essential for the transcription of the TGF-beta2 gene in U87MG cells. Gel mobility analysis determined that activating transcription factor-1, and possibly cAMP-responsive element binding protein, binds to the CRE/ATF site, and upsteam stimulatory factor (USF) 1 and USF2 bind to the E-box motif. Interestingly, expression of a dominant negative USF protein down-regulates TGF-beta2 activity by 80-95% in glioblastoma cells. We conclude that the binding of transcription factors, in particular the USF proteins, to the TGF-beta2 promoter is essential for its expression and possibly its up-regulation in glioblastomas.
The expression of transforming growth factor- beta 2 (TGF- beta 2) appears to play a strong role in the establishment and progression of glial tumors. In particular, elevated expression of TGF- beta ...2 appears to be responsible for the impaired cell-mediated immunity often observed in patients with a glioblastoma. This study examined the regulation of the TGF- beta 2 at the transcriptional level in the U87MG glioblastoma cell line. We demonstrate that a cAMP response element/activating transcription factor (CRE/ATF) site and an E-box motif located just upstream of the transcription start site are essential for the transcription of the TGF- beta 2 gene in U87MG cells. Gel mobility analysis determined that activating transcription factor-1, and possibly cAMP-responsive element binding protein, binds to the CRE/ATF site, and upsteam stimulatory factor (USF) 1 and USF2 bind to the E-box motif. Interestingly, expression of a dominant negative USF protein down-regulates TGF- beta 2 activity by 80-95% in glioblastoma cells. We conclude that the binding of transcription factors, in particular the USF proteins, to the TGF- beta 2 promoter is essential for its expression and possibly its up-regulation in glioblastomas.
A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the
inner leaflet of the plasma membrane of resting mammalian cells. We have ...shown previously that PS becomes exposed on the surface
of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed.
3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice.
A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4
and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein β-2-glycoprotein
1 (β2GP1). β2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show
that 3G4 enhances binding of β2GP1 to EC induced to expose PS. We also show that divalent 3G4-β2GP1 complexes are required
for enhanced binding, since 3G4 Fabâ² fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial
dimeric β2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by
dimerization of β2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of β2GP1 for anionic
phospholipids through formation of multivalent 3G4-β2GP1 complexes.
Gene transcription is a highly complex process that is strictly regulated to ensure proper spatial and temporal gene expression. To ensure proper expression, and prevent aberrant transcription, many, ...if not most, genes are regulated by enhancer elements. In this regard, transcription of the fibroblast growth factor-4 (FGF-4) gene during early development is controlled by a powerful distal enhancer located 3 kb downstream of the transcription start site within the 3′ untranslated region of the gene. Previous studies have shown that FGF-4 enhancer function is mediated by at least three critical positive cis-regulatory elements: an HMG, a POU, and a GT-box motif, which bind the transcription factors Sox-2, Oct-3, and Sp1/Sp3, respectively. In this dissertation, a second essential HMG motif is identified within the FGF-4 enhancer that binds the transcription factor Sox-2. Furthermore, it is shown using embryonic stem cells containing targeted disruptions of both Sp1 alleles, that the transcription factor Sp1 is not required for expression of FGF-4 transcripts in vivo. In addition, the transcription factor Sp3 is identified as a more potent mediator of the enhancer GT-box than Sp1, suggesting that Sp3 likely mediates that effects of the enhancer GT-box motif in vivo. Lastly, it is shown that a precise spatial arrangement of the new HMG motif, relative to the previously identified enhancer cis-regulatory elements, is required for optimal enhancer function. This is important, because enhancer mediated activation of many genes is accomplished by an intricate array of enhancer cis-regulatory elements that direct the assembly of a gene-specific activation complex known as an “enhanceosome.” Based on findings presented in this dissertation, and work published earlier demonstrating that the previously identified HMG and POU motifs of the FGF-4 enhancer function cooperatively and require a strict spatial arrangement, it is proposed that the FGF-4 enhancer functions, in part, as an enhanceosome. Thus, the work presented in this dissertation advances our understanding of FGF-4 gene regulation and the function of distal enhancer elements.
Outpatient stroke rehabilitation is often lengthy and expensive due to patients' lack of functional use of the impaired arm outside of the clinic caused by "learned non-use." Learned non-use is ...detrimental to stroke recovery, often resulting in chronic disability. To overcome learned non-use, a wearable "personal assistant" solution is proposed that employs ubiquitous cueing to stimulate patient use of the paretic arm while outside of therapy sessions. A pilot user study is presented that evaluated stroke survivors' tolerance and acceptance of cueing, and the usability of the proposed implementation.
Outpatient stroke rehabilitation is often lengthy and expensive due to patients' lack of functional use of the impaired arm outside of the clinic caused by "learned non-use." Learned non-use is ...detrimental to stroke recovery, often resulting in chronic disability. To overcome learned non-use, a wearable "personal assistant" solution is proposed that employs ubiquitous cueing to stimulate patient use of the paretic arm while outside of therapy sessions. A pilot user study is presented that evaluated stroke survivors' tolerance and acceptance of cueing, and the usability of the proposed implementation.
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