Identification and characterization of the ING1 and ING2 tumor suppressors during thyroid hormone-dependent tadpole metamorphosis




Wagner, Mary Jeannette

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The ING (INhibitor of Growth) tumor suppressor genes are conserved from yeast to humans and are implicated in several processes important to cell proliferation and apoptosis. ING proteins contain a plant homeodomain (PHD) finger that suggests these proteins may modulate transcription factor-mediated pathways. Little is known about the mechanism of action of INGs, especially in the context of normal development. The ING family of proteins includes at least five different genes, ING1-ING5, with evidence for alternate promoter usage and splicing that generate multiple isoforms. To elucidate the role of ING in different tissues to modulate function, I used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates apoptosis, proliferation, and remodeling in the tail, hindlimb, and brain, respectively. I discovered seven ING1 and three ING2 transcript variants in Xenopus laevis and investigated their expression patterns. High expression levels of most variants were found in adult brain, testis, and eye. During natural metamorphosis or precocious metamorphosis induced by treating tadpoles with exogenous TH, ING1 and ING2 transcript variant levels were differentially regulated in a tissue-specific manner. Some variant levels increased with the induction of apoptosis of the tail, while levels of the same variants decreased upon induction of proliferation and differentiation in the hindlimb. Although levels of all INC variants were relatively high in whole brain, they did not change during metamorphosis or TH treatment. Given that ING has previously been shown to modulate apoptosis, it is likely that upregulation of specific isoforms may contribute to the tissue-specific TH-mediated response in the tail, and that downregulation facilitates proliferation of the hindlimb. To further investigate the hypothesis that ING is regulated by TH, an analysis of 1NG1 and ING2 genomic sequences was carried out. Promoter sequences for each variant were determined and putative thyroid hormone response elements (TREs) located. To test whether thyroid hormone receptors associate with these elements, chromatin immunoprecipitations (ChIP) assays were done on tail homogenates from premetamorphic tadpoles treated with TH or vehicle control. Both thyroid hormone receptor α (TRα) and thyroid hormone receptor β (TRβ) differentially associate with ING1 and ING2 promoter regions. TR association increased significantly on promoters for ING variant transcripts that increase upon TH treatment, and decreased significantly on promoters for ING variant transcripts that decrease upon TH treatment. ChIPs also showed that ING associates with TH-regulated promoters including TRβ, TH-Responsive Basic Leucine Zipper Transcription Factor (TH/bZIP), ING1 and ING2. Furthermore, TR and ING were shown to co-immunoprecipitate with both purified proteins and using total tail homogenates from metamorphic tadpoles. The antibodies used for these experiments were made against Xenopus TRβ and ING2 and were characterized as part of this thesis. Bioinformatics revealed that TREs are present in promoters of ING genes for other species including human, mouse, and a related frog species, Xenopus tropicalis; therefore, it is likely that modulation by TH is a conserved mechanism of ING regulation. These data suggest that there may be antagonistic regulation of ING transcript variants by TH that correlates with tissue fate. TRs associate with ING promoters, and ING is associated with TR-regulated promoters. Moreover, TR and ING proteins co-immunoprecipitate. It is therefore likely that TR and ING are co-regulators of gene expression during TH-dependent tadpole metamorphosis. This thesis contributes to the understanding of ING which is relevant to elucidating many disease states, as well as being critical in understanding the role of this tumor suppressor in the context of TH regulation and normal development.



amphibians, metamorphosis, thyroid hormones