GLIS3 regulates transcription of thyroid hormone biosynthetic genes in coordination with other thyroid transcription factors.

BACKGROUND: Loss of the transcription factor GLI-Similar 3 (GLIS3) function causes congenital hypothyroidism (CH) in both humans and mice due to decreased expression of several thyroid hormone (TH) biosynthetic genes in thyroid follicular cells. Whether and to what extent, GLIS3 regulates thyroid gene transcription in coordination with other thyroid transcriptional factors (TFs), such as PAX8, NKX2.1 and FOXE1, is poorly understood. METHODS: PAX8, NKX2.1, and FOXE1 ChIP-Seq analysis with mouse thyroid glands and rat thyrocyte PCCl3 cells was performed and compared to that of GLIS3 to analyze the co-regulation of gene transcription in thyroid follicular cells by these TFs. RESULTS: Analysis of the PAX8, NKX2.1, and FOXE1 cistromes identified extensive overlaps between these TF binding loci and those of GLIS3 indicating that GLIS3 shares many of the same regulatory regions with PAX8, NKX2.1, and FOXE1, particularly in genes associated with TH biosynthesis, induced by thyroid stimulating hormone (TSH), and suppressed in Glis3KO thyroid glands, including Slc5a5 (Nis), Slc26a4, Cdh16, and Adm2. ChIP-QPCR analysis showed that loss of GLIS3 did not significantly affect PAX8 or NKX2.1 binding and did not cause major alterations in H3K4me3 and H3K27me3 epigenetic signals. CONCLUSIONS: Our study indicates that GLIS3 regulates transcription of TH biosynthetic and TSH-inducible genes in thyroid follicular cells in coordination with PAX8, NKX2.1, and FOXE1 by binding within the same regulatory hub. GLIS3 does not cause major changes in chromatin structure at these common regulatory regions. GLIS3 may induce transcriptional activation by enhancing the interaction of these regulatory regions with other enhancers and/or RNA Polymerase II (Pol II) complexes.

Comparison of clonogenic cell survival and DNA damage induced by 188Re and X-rays in rat thyroid cells.

AIM: Ionizing radiation produces DNA lesions among which DNA double strand breaks (DSB) are the most critical events. Radiation of various energy types might differ in their biological effectiveness. Here, we compared cell survival and DNA damage induced by 188Re and X-rays using γH2AX foci as a measure of DSB. The correlation between survival and residual foci was also analyzed. METHODS: PCCl3 cells were irradiated with 200 kV X-rays (1.2 Gy/min) or 0.5-25 MBq/ml 188Re (1 h irradiation) achieving doses up to 10 Gy. By blocking of sodium iodide symporter (NIS) essentially extracellular activity could be guaranteed. Survival fractions (SF) were detected by colony forming assay. Initial and residual γH2AX foci (15 min and 24 h after irradiation) were assessed by immunostaining. The relationship between SF and residual radiation induced γH2AX foci (RIF) was evaluated by Spearman and Pearson correlation tests. RESULTS: We did not find significant differences between the survival curves in terms of the radiation quality. The D37 values were 4.6 Gy and 4.2 Gy for 188Re or X-ray, respectively. The initial foci numbers were in the same range for 188Re and X-ray, but higher levels of residual foci persisted after X-rays in comparison to 188Re (1 GyX-ray 6.5 ± 0.2; 1 GyRe-188 4.8 ± 0.2 RIF). Accordingly, for 188Re a higher extent of DSB repair was found. The Spearman test revealed a significant (p < 0.01) correlation between SF and residual RIF for both radiation modalities. CONCLUSION: No differences in terms of radiation were found for SF and initial foci. However, residual foci were lower for 188Re than for X-rays. A prediction of SF by residual foci should consider the properties of the radiation qualities that influence foci removal and DSB repair.

The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue.