#Regulation of Sp-Family Proteins
The research in our laboratory is focused on mechanisms of
mammalian cell growth regulation. In the
recent past our efforts have focused on the following inter-related areas of research:
(1) Regulation of the Sp-Family of Transcription Factors:
If the movie shown in the middle is not working Click hereWe have shown that trans-activation by two members of the Sp-family of transcription factors, Sp1 and Sp3, is "superactivated" by the retinoblastoma (Rb) protein in vivo. Several years ago we showed that (A) Sp3 encodes at least three distinct proteins, (B) two result from internal translational initiation, and (C) internally initiated Sp3 proteins function as potent repressors of Sp1/Sp3-mediated transcription.
Our current efforts are directed in part towards understanding the precise mechanisms involved in the "superactivation" of Sp1/Sp3 by Rb. We have also spent a great deal of time trying to understand how Sp3 isoforms produced via internal translational initiation negatively-regulate transcription. In the last year or two we co-discovered that all Sp3 isoforms are sumoylated in vivo, and we have shown that this post-translational modification is required for Sp3 to repress transcription. We have also shown that unlike many other sumoylated proteins, sumoylation of Sp3 isoforms does not alter their subcellular localization.
Most recently we have characterized the biochemical and functional properties of Sp2, a hitherto poorly characterized member of the Sp-family of proteins. Our studies have shown that, unlike other Sp-family members, Sp2 is a relatively weak trans-activator in vivo and that at least two distinct mechanisms negatively regulate Sp2-mediated trans-activation. Our current efforts are geared towards a better understanding of these two mechanisms as well as the signaling pathways that govern their activity.
Additionally, we are expending considerable effort to study the cell biology of the Sp-family of transcription factors and Nkx3.1 (see below) using indirect immunofluorescence as well as via the creation of GFP-fusion proteins and their visualization in live cells using time-lapse confocal microscopy. These studies have led to a number of novel findings as well as many pretty pictures and cell movies (roll your mouse over the movie below to activate).
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(2) Nkx3.1:
Nkx3.1 is a homeodomain-containing transcription factor that plays a key role in the formation of the human and mouse prostate gland. Loss of Nkx3.1, due to mutation or deletion, is also associated with prostate tumorigenesis. In recent years we have shown that Nkx3.1 physically interacts with Sp-family members and negatively regulates their capacity to stimulate transcription. Our current efforts are geared towards revealing the precise mechanism whereby Nkx3.1 blocks Sp protein function as well as defining post-translational mechanisms that govern Nkx3.1 function.
(3) Rb-Binding Proteins:
We have identified nine cellular proteins that specifically interact with the amino-terminal end of human and mouse Rb proteins in vitro. We characterized a series of cDNAs cloned via a yeast "two-hybrid" screen and showed that one encodes MCM7, a key regulator of the initiation of DNA replication. Using a variety of biochemical and functional assays we have shown that (A) the amino-termini of Rb and two Rb-related proteins, p107 and p130, bind MCM7 in vitro and in vivo and (B) that the amino-termini of Rb and p130 can block DNA replication in vitro in an MCM7-dependent fashion. These results provided the first evidence that DNA replication is directly regulated by tumor-suppressor genes.
(4) Novel Dimerization Partners of E2F/DP Proteins:
We have carefully characterized sites of phosphorylation by cyclin/cdk kinases of DP-2, an E2F dimerization partner that we co-discovered several years ago. Moreover, using degenerate oligonucleotides, the PCR, and a sensitive in vitro protein/DNA-binding assay we have defined a series of preferred DNA-binding sites for a variety of E2F/DP and Rb/E2F/DP complexes. These studies have shown that E2F complexes prefer particular E2F-binding sites, that E2F and DP proteins play a role in the selection of such sites, that Rb significantly alters the DNA-binding site specificity of E2F/DP complexes, and that E2F sites selected in vitro by various E2F complexes exhibit distinct patterns of cell cycle-regulated transcription in vivo.
Publications (1996/2006):
Sterner, J.M., Tao Y., Kennett, S.B., Kim, H.G., Horowitz, J.M. The amino-terminus of the retinoblastoma (Rb) protein associates with a cdk-like kinase via Rb amino acids required for growth suppression. Cell Growth Differ. 7:53-64, 1996.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Rogers, K.T., Higgins, P.D.R., Milla, M., Phillips, R.S., Horowitz, J.M. DP-2, a heterodimeric partner of E2F: Identification and characterization of DP-2 proteins expressed in vivo. Proc. Natl. Acad. Sci. U.S.A. 93:7594-7599, 1996.
Or, follow this link to read the paper online. If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Cuevo, R.S., Garrett, S., Horowitz, J.M. Detection and functional characterization of p180, a novel cell-cycle regulated yeast transcription factor that binds retinoblastoma control elements (RCEs). J. Biol. Chem. 272:3813-3822, 1997.
Or, follow this link to read the paper online. If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Kennett, S.B., Udvadia, A.J., Horowitz, J.M. Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucl. Acids Res. 25:3110-3117, 1997.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Tao, Y., Kassatly, R.F., Cress, D., and Horowitz, J.M. Subunit composition determines E2F-binding site specificity. Mol. Cell. Biol. 17:6994-7007, 1997.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Sterner, J.M., Dew-Knight, S., Musahl, C., Kornbluth, S., and Horowitz, J.M. Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7. Mol. Cell. Biol. 18:2748-2757,1998.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Lee, R.J., Albanese, C., Fu, M., D'Amico, M., Lin, B.G., Watanabe, G.K., Haines III, P.M. Siegel, Muller, W.J., Yarden, Y., Horowitz, J.M., Hung, M.-C., and Pestell, R.G. Cyclin D1 is required for transformation and is transcriptionally activated by Neu/erbB-2. Mol. Cell. Biol. 20:672-683, 2000.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Baek, S.J., Horowitz, J.M., and Eling, T.E. Molecular cloning and characterization of human NSAID activated gene, NAG-1, promoter: Basal transcription is mediated by Sp1 and Sp3. J. Biol. Chem. 276: 33384-33392, 2001.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Kennett, S.B., Moorefield, K.S., and Horowitz, J.M. Sp3 represses gene expression via the titration of promoter-specific transcription factors. J. Biol. Chem. 277: 9780-9789, 2002.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Moorefield, K.S., Fry, S.J. and Horowitz, J.M. Sp2 DNA-binding activity and trans-activation are negatively regulated in mammalian cells. J. Biol. Chem. 279: 13911-13924, 2004.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Spengler, M.L., Kennett, S.B., Moorefield, K.S., Simmons, S.O., Brattain, M.G., and Horowitz, J.M. Sumoylation of internally-initiated Sp3 isoforms regulates transcriptional repression via a Trichostatin A-insensitive mechanism. Cell. Signal. 17:153-166, 2005.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Simmons, S.O. and Horowitz, J.M. Nkx3.1 binds and negatively regulates the transcriptional activity of Sp-family members in prostate-derived cells. Biochemical J. 393:397-409, 2006.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Moorefield, K.S., Nichols, T.D., Simmons, S.O., Cathcart, C., and Horowitz, J.M. Sp2 localizes to sub-nuclear foci associated with the nuclear matrix. Mol. Biol. Cell 17: 1711-1722, 2006.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Rong,
Y., Hu, F., Huang, R.P., Mackman, N., Horowitz , J.M., Jensen, R.L., Durden, D.L.,
Van Meir, E.G., Brat, D.J. Egr-1 regulates hypoxia-induced expression of tissue
factor in glioblastoma through HIF-1α independent mechanisms.
Cancer
Res. 66:
7067-7074, 2006.
If you have the Adobe Acrobat Reader installed on your computer, you can use this link to read the paper directly.
Correspondence:
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