Abstract
Recent studies show that Arf, a bona fide tumor suppressor, also plays an essential role during mouse eye development. Tgfβ is required for Arf promoter activation in developing mouse eyes, and its capacity to induce Arf depends on Smads 2/3 as well as p38 Mapk. Substantial delay between activation of these pathways and increased Arf transcription imply that changes in the binding of additional transcription factors help orchestrate changes in Arf expression. Focusing on proteins with putative DNA binding elements near the mouse Arf transcription start, we now show that Tgfβ induction of this gene correlated with decreased expression and DNA binding of C/ebpβ to the proximal Arf promoter. Ectopic expression of C/ebpβ in mouse embryo fibroblasts (MEFs) blocked Arf induction by Tgfβ. Although basal levels of Arf mRNA were increased by C/ebpβ loss in MEFs and in the developing eye, Tgfβ was still able to increase Arf, indicating that derepression was not the sole factor. Chromatin immunoprecipitation (ChIP) assay showed increased Sp1 binding to the Arf promotor at 24 and 48 hours after Tgfβ treatment, at which time points Arf expression was significantly induced by Tgfβ. Chemical inhibition of Sp1 and its knockdown by RNA interference blocked Arf induction by Tgfβ in MEFs. In summary, our results indicate that C/ebpβ and Sp1 are negative and positive Arf regulators that are influenced by Tgfβ.
Highlights
Arf, a bona fide mammalian tumor suppressor gene transcribed from the Cdkn2a locus, encodes p19Arf in an alternative reading frame when compared to p16Ink4a, the first gene found at this chromosomal locus [1]
Recognizing the substantial delay between Smad binding to the Arf promoter and increased synthesis of Arf primary transcript [22], we considered potential roles for other transcription factors whose function might be influenced by Tgfb
Utilizing chromatin immunoprecipitation (ChIP), we demonstrated that C/ebpb was bound to this region in cultured mouse embryo fibroblasts (MEFs) at passage 3 (YZ and SXS, unpublished data)
Summary
A bona fide mammalian tumor suppressor gene transcribed from the Cdkn2a locus, encodes p19Arf in an alternative reading frame when compared to p16Ink4a, the first gene found at this chromosomal locus [1]. Arf coding sequence can be deleted in mouse and human tumors, in a substantial number the gene is intact but silenced alone or together with INK4A [4,5]. Global silencing of its expression is mediated by chromatin remodeling proteins such as Bmi since the expression of both Arf and Ink4a increase when Bmi is deleted in mouse models [8]. In this regard, a long non-coding RNA (ANRIL), transcribed anti-parallel to human ARF and INK4a (and the INK4b gene lying further 59 of ARF/INK4a) [9] acts in cis to foster CBX7 binding to this region in cultured human PC3 cells [10]. It is important to note that many of these conclusions stem from highly tractable cell culture models, but the in vivo relevance is less clear in most cases
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