Sp1, a transcription factor that regulates expression of a wide array of essential genes, contains two SQ/TQ cluster domains, which are characteristic of ATM kinase substrates. ATM substrates are transducers and effectors of the DNA damage response, which involves sensing damage, checkpoint activation, DNA repair, and/or apoptosis. A role for Sp1 in the DNA damage response is supported by our findings: Activation of ATM induces Sp1 phosphorylation with kinetics similar to H2AX; inhibition of ATM activity blocks Sp1 phosphorylation; depletion of Sp1 sensitizes cells to DNA damage and increases the frequency of double strand breaks. We have identified serine 101 as a critical site phosphorylated by ATM; Sp1 with serine 101 mutated to alanine (S101A) is not significantly phosphorylated in response to damage and cannot restore increased sensitivity to DNA damage of cells depleted of Sp1. Together, these data show that Sp1 is a novel ATM substrate that plays a role in the cellular response to DNA damage. (Mol Cancer Res 2007;5(12):1319–30)

Purpose: Proangiogenic protein vascular endothelial growth factor A (VEGF-A) contributes significantly to retinal lesions and neovascularization in diabetic retinopathy (DR). In pre-clinical DR, hyperglycemia can upregulate VEGF-A in retinal cells. The VEGF-A promoter is responsive to the transcription factor specificity protein 1 (Sp1). The O-GlcNAc modification is driven by glucose concentration and has a profound effect on Sp1 activity. This study investigated the effects of hyperglycemia on Sp1-mediated expression of VEGF-A in the retinal endothelium and pigment epithelium. Methods: Hyperglycemia-exposed ARPE-19 (human retinal pigment epithelial cells) and TR-iBRB (rat retinal microendothelial cells) were assayed for levels of VEGF-A by qRT-PCR, Western blot, and ELISA. Small molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) were used to manipulate O-GlcNAc levels. VEGF-A protein and transcript were measured in cells depleted of OGT or Sp1 by shRNA. The proximal VEGF-A promoter was analyzed for glucose sensitivity by luciferase assay. Chromatin immunoprecipitation (ChIP) was used to assess Sp1 occupancy on the VEGF-A promoter. Results: Hyperglycemia increased VEGF-A promoter activity and upregulated VEGF-A transcript and protein. Elevation of O-GlcNAc by OGA inhibitors was sufficient to increase VEGF-A. OGT inhibition abrogated glucose-driven VEGF-A. Cellular depletion of OGT or Sp1 by shRNA significantly abrogated glucose-induced changes in VEGF-A. ChIP analysis showed that hyperglycemia significantly increased binding of Sp1 to the VEGF-A promoter. Conclusions: Hyperglycemia-driven VEGF-A production is mediated by elevated O-GlcNAc modification of the Sp1 transcription factor. This mechanism may be significant in the pathogenesis of pre-clinical diabetic retinopathy through VEGF-A upregulation.

Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.

Human cytomegalovirus (HCMV) immediate-early protein IE1/IE72 is involved in undermining many cellular processes including cell cycle regulation, apoptosis, nuclear architecture, and gene expression. The multifunctional nature of IE72 suggests that posttranslational modifications may modulate its activities. IE72 is a phosphoprotein and has intrinsic kinase activity (S. Pajovic, E. L. Wong, A. R. Black, and J. C. Azizkhan, Mol. Cell. Biol. 17:6459-6464, 1997). We now demonstrate that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1). SUMO-1 is an 11.5-kDa protein that is conjugated to multiple proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. A covalently modified protein migrating at ∼92 kDa, which is stabilized by a SUMO-1 hydrolase inhibitor, is revealed by Western blotting with anti-IE72 of lysates from cells infected with HCMV or cells expressing IE72. SUMO modification of IE72 was confirmed by immunoprecipitation with anti-IE72 and anti-SUMO-1 followed by Western blotting with anti-SUMO-1 and anti-IE72, respectively. Lysine 450 is within a sumoylation consensus site (I,V,L)KXE; changing lysine 450 to arginine by point mutation abolishes SUMO-1 modification of IE72. Inhibition of protein phosphatase 1 and 2A, which increases the phosphorylation of IE72, suppresses the formation of SUMO-1-IE72 conjugates. Both wild-type IE72 and IE72_K450R localize to nuclear PML oncogenic domains and disrupt them. Studies of protein stability, transactivation, and complementation of IE72-deficient HCMV (CR208) have revealed no significant differences between wild-type IE72 and IE72_K450R.

Herpes keratitis (HK) is the leading cause of cornea-derived and infection-associated blindness in the developed world. Despite the availability of effective antivirals, some patients develop refractory disease, drug-resistant infection, and topical toxicity. A nonpharmaceutical treatment modality may offer a unique advantage in the management of such cases. This study investigated the antiviral effect of nonthermal dielectric barrier discharge (DBD) plasma, a partially ionized gas that can be applied to organic substances to produce various biological effects. Human corneal epithelial cells and explanted corneas were infected with herpes simplex virus type 1 (HSV-1) and exposed to culture medium treated with nonthermal DBD plasma. The extent of infection was measured by plaque assay, quantitative PCR, and Western blot. Corneal toxicity assessment was performed with fluorescein staining, histologic examination, and 8-OHdG detection. Application of DBD plasma–treated medium to human corneal epithelial cells and explanted corneas produced a dose-dependent reduction of the cytopathic effect, viral genome replication, and the overall production of infectious viral progeny. Toxicity studies showed lack of detrimental effects in explanted human corneas. Nonthermal DBD plasma substantially suppresses corneal HSV-1 infection in vitro and ex vivo without causing pronounced toxicity. Nonthermal plasma is a versatile tool that holds great biomedical potential for ophthalmology, where it is being investigated for wound healing and sterilization and is already in use for ocular microsurgery. The anti-HSV-1 activity of DBD plasma demonstrated here could be directly translated to the clinic for use against drug-resistant herpes keratitis.