My laboratory has a long standing interest in regulation of gene expression and cell proliferation as they relate to cancer. Our work has focused on the transcriptional regulation of genes that lack a TATA element in their promoter, which was once thought to be a canonical feature. Genes regulated by these so-called TATA-less promoters include genes involved in regulation of many metabolic processes, DNA replication, DNA repair, and apoptosis, as well as growth factors and their receptors, oncogenes and tumor suppressors. Promoters for these genes are GC-rich and most contain multiple sites that bind the transcription factor Sp1. We and others demonstrated that in TATA-less promoters, Sp1 functions to control transcription initiation and recruit the general transcription machinery through protein-protein interactions with a component of the TBP-containing general transcription factor, TFIID. Although considered a “general” transcription factor, regulation of transcription of a large number of genes in response to a wide array of signals has been ascribed to Sp1. Sp1 is post-translationally modified by phosphorylation, acetylation, O-linked glycosylation, sumoylation, ubiquitylation, and methylation. These modifications affect not only DNA binding, but also Sp1 activity and interactions with other factors. Our work has largely focused on regulation of Sp1 activity through modulation of phosphorylation, with some work and significant interests related to acetylation, sumoylation and glycosylation.
Several years ago, a graduate student in the lab discovered that Sp1 is significantly phosphorylated in response to DNA damage. Sp1 is phosphorylated by several different kinases at one or more of its 96 Ser residues. Clearly, phosphorylation at different sites by different kinases differentially modulates its activity in response to different signals. Much of our current work is focused on phosphorylation in response to DNA damage and the role of Sp1 in the cellular response to damage. Eleven of the 96 Ser residues in Sp1 are SQ sequences clustered in the glutamine-rich transactivation domains; S/TQ cluster domains (SCDs) are characteristic of proteins phosphorylated by ATM/ATR in response to DNA damage. We have found that Sp1 is phosphorylated by ATM on several Ser residues in response to reactive oxygen species (ROS) generated by DNA damage and that its phosphorylation is involved in the increased sensitivity to DNA damage observed in cells depleted of Sp1. Phosphorylation on S101 is required for additional phosphorylation, i.e. its phosphorylation primes for additional phosphorylation, and we have made an antibody that specifically detects Sp1 phosphorylated on S101 in cells subjected to DNA damage. We have shown by immunofluorescence/confocal microscopy and chromatin immunoprecipitation that phospho-Sp1 is localized to sites of DNA damage and that its phosphorylation is dependent on the presence of Nbs1, a key component of the MRN complex that recruits ATM to DSB sites. We have also shown that Sp1 is phosphorylated in response to UV, and although S101 is phosphorylated by ATM (and not by ATR) after UV, phosphorylation at S101 is not a priming phosphorylation. We are also studying the role of Sp1 in the induction of apoptosis after DNA damage. Sp1 is preferentially degraded by caspases at higher levels of damage, particularly after UV. Degradation of Sp1 is associated with induction of apoptosis, and blocking caspase-mediated cleavage (by mutation of the specific aspartic acid cleaved by caspase) protects cells from damage-induced apoptosis.
Having demonstrated that mutation of S101 precludes additional phosphorylation in response to DNA damage, we would like to understand at a biochemical level how the phosphorylation of this residue apparently primes the protein for additional phosphorylation and how phosphorylation affects its activity in the DNA damage response and in transcription. Microarray studies are underway to look at Sp1-dependent changes in gene expression in response to DNA damage
Current studies are directed at demonstrating the mechanism whereby Sp1 modulates the cellular response to DNA damage, including studies of: activation/recruitment of downstream effectors to DNA damage sites, checkpoint activation, chromatin remodeling, DNA repair, apoptosis induction, and transcription modulation. We are also trying to develop our phospho-specific antibody, which is a very sensitive indicator of DNA damage, as a marker that could be used to guide treatment of patients with radiation or chemotherapy and/or to detect environmental exposure to DNA damage. This is particularly significant because 20% of people do not express H2AX, the only damage marker in current use. We are developing our antibody, γ Sp1101 as a diagnostic tool to measure DNA damage in peripheral blood of patients subjected to irradiation and/or chemotherapy.
There are several studies underway and planned to establish the clinical significance of our findings. These include identification of Sp1 mutations in tumors. Sp1 overexpression has been reported in several cancers and some studies have suggested that overexpression is an indicator of poor prognosis; however, there are no reports of specific mutations in Sp1 in tumors.
Sp1 has also been implicated in neurodegenerative diseases, particularly Alzheimer’s and Huntingtin’s disease; however no one has figured out how it is involved (regulates tau, APP and COX-2; stimulated by IL1β (inflammation). Sp1 is acetylated as well as phosphorylated in response to ROS and blocking its acetylation has been linked to the neuroprotective effect of compounds like TSA. Sp1 is increased in AD brains. We are performing experiments to explore the mechanism by which Sp1 is neuroprotecti ve.
Sp1 was the first transcription factor shown to be O-glycosylated; however, the sites of glycosylation in response to specific signals have not been thoroughly mapped and the functional significance of glycosylation remains a mystery. We are exploring the function of Sp1 glycosylation by mapping sites of O-glycosylation, and the effect of agents that block glycosylation on Sp1-dependent functions.
Other projects: In collaboration with Gary Friedman and Yuri Gogotsi (College of Engineering), we are studying the effects of non-thermal plasma on cells. Plasma is comprised of electrically charged molecules, electrons as well as some highly active neutral molecules (electronically excited atoms and radicals) that can be produced through application of a strong electric field. In this work we employ electrodes with a dielectric barrier to produce plasma whose average temperature is close to room temperature. We have shown that when applied to a solution, non-thermal plasma results in the generation of reactive oxygen species in a dose-dependent and highly controllable manner (even in single cells). The goal is to develop cold plasma for utilization in sterilization of surfaces, particularly wounds (if selectivity between effects on bacteria and cells can be achieved) and to induce apoptosis in cancer cells by local administration. We are characterizing the reactive oxygen species that are produced and their effects on DNA.
Selected Publications
Black, A.R., Jensen, D.E., Lin, S.-Y., and Azizkhan, J.C. Growth/Cell cycle regulation of Sp1 phosphorylation. J. Biol. Chem. 274:1207-1215, 1999.
Ryu, H., Lee J., Olofsson, B.A., Mwidau, A., Dedeoglu, A., Escudero, M., Flemington, E., Azizkhan-Clifford, J., Ferrante, R.J., Ratan, R.R. Histone deacetylase inhibitors prevent oxidative neuronal death independent of expanded polyglutamine repeats via an Sp1-dependent pathway. Proc. National Acad. Sciences 100(7):4281-4286, 2003.
Mitra J, Enders GH, Azizkhan-Clifford J, Lengel KL. Dual regulation of the anaphase promoting complex in human cells by cyclin A-Cdk2 and cyclin A-Cdk1 complexes. Cell Cycle. 2006 Mar;5(6):661-6, 2006.
Olofsson, B., Kelly, C.M., Kim, J., Hornsby, S., and Azizkhan-Clifford, J. Phosphorylation of Sp1 in Response to DNA Damage by Ataxia Telangiectasia-Mutated Kinase, Molecular Cancer Research 5(12):1319-1330, 2007.
Kelly, C.M., Olofsson, B., Emrich, J., Greenberg, R.A. and Azizkhan-Clifford, J. Sp1 Phosphorylation by ATM and Recruitment to Sites of DNA Double Strand Breaks is Dependent on Interaction with Nbs1, Submitted to Molecular Cell.
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