Abstract
BackgroundINhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action.Methodology/Principal FindingsTadpoles expressing a Xenopus ING2 transgene (TransING2) were significantly smaller than tadpoles not expressing the transgene (TransGFP). When exposed to 10 nM 3,5,3′-triiodothyronine (T3), premetamorphic TransING2 tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T3-treated TransGFP tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor β (TRβ) and TH/bZIP transcript levels in TransING2 tadpole tails compared to TransGFP tadpoles while TRα mRNAs were unaffected. In contrast, no difference in TRα, TRβ or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between TransING2 and TransGFP tadpoles. All of these transcripts, except for TRα mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T3-induced TRβ gene promoter activity. Examination of endogenous T3-responsive promoters (TRβ and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T3-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals.Conclusions/SignificanceWe show for the first time that ING proteins modulate TH-dependent responses, thus revealing a novel role for ING proteins in hormone signaling. This has important implications for understanding hormone influenced disease states and suggests that the induction of ING proteins may facilitate TR function during metamorphosis in a tissue-specific manner.
Highlights
INhibitor of Growth (ING) proteins are implicated in the control of several key cellular processes including proliferation, apoptosis, DNA repair, senescence, and drug resistance; their transcript levels are often reduced in cancer cells [1,2,3]
The basic plasmid construct contains a Xenopus cardiac actin promoter (XCAR)-driven green fluorescent protein (GFP) that enables the rapid screening of embryos for positive transgenics by virtue of detectable GFP expression in mesoderm
Given that regulation of mRNA expression is a major mechanism of thyroid hormone (TH) action and that ING proteins are known to associate with chromatin and modulate gene promoter activity, we examined whether Inhibitor of growth 2 (ING2) overexpression affects the TH-mediated developmental program in transgenic animals
Summary
ING proteins are implicated in the control of several key cellular processes including proliferation, apoptosis, DNA repair, senescence, and drug resistance; their transcript levels are often reduced in cancer cells [1,2,3]. The latter property likely results from epigenetic regulation of the ING genes through events such as DNA hypermethylation, as mutations in these genes are rare [4]. The present study investigates the possibility that ING proteins modulate TH action
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