Sp100 Interacts with ETS-1 and Stimulates Its Transcriptional Activity

Abstract

The cell nucleus is highly organized into distinct domains that spatially separate physiological processes. One of these domains, the Sp100-promyelocytic leukemia protein nuclear body (NB), is implicated in pathological processes, such as cancer and viral infection, yet its functions remain poorly understood. We show here that Sp100 interacts physically and functionally with ETS-1 and that NB morphology is affected by ETS-1. ETS-1 is a member of the ets family of transcription factors, which are key mediators of physiological and pathological processes. We have found that Sp100 interacts with two regions of ETS-1 (domains A؉B and D؉E؉F). ETS-1 alters NBs while remaining localized throughout the nucleus, apparently by recruitment of the core component Sp100 away from the NBs. Sp100 strongly increases ETS-1 activation of natural and ets-focused promoters, through a mechanism involving the activation (C) domain of ETS-1 in addition to the interaction domains. Sp100 acts as a novel coactivator that potentiates the activator function of ETS-1. Our results provide an important new connection between nuclear structures and an important regulator of gene expression.Physiological processes, such as transcription, DNA replications and pre-mRNA splicing, take place in defined nuclear domains. A part of the global nuclear architecture is the Sp100-PML nuclear body (NB, also called nuclear domain 10, and promyelocytic leukemia protein [PML] oncogenic domain). NBs appear to be involved in the pathogenesis of human diseases, including acute promyelocytic leukemia and viral infection, and appear to have physiological roles in the control of cell growth, differentiation and apoptosis (for reviews, see references 24, 38, 41, 56, and 64). NBs were initially identified by electron microscopy. They are immunologically defined as containing two major components, Sp100 and PML. Sp100 was first characterized as an antigen reactive with antibodies from patents with autoimmune disorders (67). PML was identified as part of a fusion protein with retinoic acid receptor ␣ (PML-RAR␣) expressed in acute promyelocytic leukemia (for a review, see reference 38). NBs contain, in addition to their core components Sp100 and PML, transcription factors, chromosomal proteins, tumor suppressors, and proto-oncogenes (for a review, see reference 56).Mammalians cells in general contain 5 to 20 NBs, ranging in size from 0.1 to 1 m, that are spherical or torioidal in shape. NBs are highly dynamic structures. They are disrupted in acute promyelocytic leukemia cells by the PML-RAR␣ fusion protein and reorganized by the physiological ligand of RAR␣, retinoic acid. NBs are disaggregated in some neurodegenerative diseases (for a review, see reference 56), during viral infection (for a review, see reference 40), and in the cell cycle (1). NBs increase in size in response to interferons, key inducers of cellular antiproliferative and antiviral responses that upregulate PML and Sp100 (18,32,62). NB structure is established and maintained through SUMO modification of...

Ets2 and protein kinase Cepsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells

“…For example, both proteins have been shown to activate a number of genes coding for extracellular matrix-degrading proteases and, therefore, are assumed to be involved in the acquisition of cellular invasiveness ( [27] and references therein). Recently, Ets1 was found to be associated also with the regulation of the promyelocytic leukaemia protein oncogenic domains ('PODs') that seem to be deregulated in invasive tumours [56]. In the present study, combined with the results we have published previously [20], we provide evidence suggesting that these Ets factors also participate in the regulation of the expression of the (breast) tumour-promoting protein, PTHrP.…”

SP100 expression modulates ETS1 transcriptional activity and inhibits cell invasion

“…It has also been hypothesized that the nuclear body serves as a dynamic nuclear depot (Negorev et al, 2001), providing a mechanism to control the activity of the proteins that localize to the NB. Although ETS1 does colocalize with SP100 in the NB, the NB does not appear to provide a significant storage depot for ETS1 relative to total ETS1 protein, as both previous indirect immunofluorescence analysis (Wasylyk et al, 2002) and the studies described here show ETS1 is found throughout the entire nucleus. Further experimentation is needed to determine if the functional interaction between ETS1 and SP100 is geographically restricted to the NB.…”

“…SP100 has been shown to interact with heterochromatin protein 1 (HP1), which functions as a transcriptional repressor (Seeler et al, 1998), and SP100 acts as repressor of transcription when tethered to DNA (Lehming et al, 1998;Seeler et al, 1998;Bloch et al, 1999). However, SP100 and ETS1 have recently been reported to coactivate the MMP3 (stromelysin) promoter as well as a heterologous promoter containing multimerized EBS (Wasylyk et al, 2002). Although no direct evidence was provided, it was proposed that ETS1 and SP100 are able to form a complex on the EBS present on the MMP3 promoter.…”

“…This Relative gene expression for each gene in the experimental Ad-SP100 and control Ad-GFP-infected cells was normalized to the expression of S26, and the relative gene expression for cells infected with the Ad-SP100 was divided by the relative gene expression for cells infected with the control Ad-GFP to determine the ratio of SP100/GFP gene expression for MMP1, uPA and uPAR, depicted as percent expression SP100/GFP interaction was first identified in a yeast two-hybrid screen ( Figure 1) and was further confirmed by coimmunoprecipitation and colocalization experiments (Figures 1 and 2), indicating that SP100 and ETS1 physically associate in vitro and in vivo. A previous report demonstrated that SP100 interacts with ETS1 at both the amino and the carboxy terminus of ETS1 (Wasylyk et al, 2002), which contain the PNT domain and the ETS (DNA-binding) domain, respectively. Since the ETS and PNT domains are the most conserved regions in the Ets family (Ghysdael and Boureux, 1997; Graves and Petersen, 1998), we predicted that SP100 might interact with other Ets family members.…”

Ets1 is an effector of protein kinase Cα in cancer cells

“…Another gene, SP100, has been decribed to be a modulator of Ets1 activity (Wasylyk et al, 2002;Yordy et al, 2004). SP100 colocalizes with PML in nuclear bodies (Salomoni and Pandolfi, 2002) whose number can be regulated by Ets1 (Wasylyk et al, 2002;Yordy et al, 2004).…”