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

Abstract

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.

Keywords: FOXE1; GLIS3; Gene transcription; NIS; NKX2.1; PAX8; PCCl3; TSH; Thyroid follicular cells; Thyroid hormone biosynthesis.

Fig. 1

Global analysis of GLIS3, NKX2.1, and PAX8 genomic binding in mouse thyroid glands. A–C HOMER analysis, heatmap, and ChIP-Seq read density of GLIS3 (A), NKX2.1 (B), and PAX8 (C) binding data. Heatmap of the 2 kb region is centered on each of the binding peaks identified. D Genomic context of the GLIS3, NKX2.1, and PAX8 peaks within the whole mouse genome (mm10)

Fig. 2

GLIS3, NKX2.1 and PAX8 binding to the mouse thyroid gland genome partially overlap. A The number of collapsed binding regions that are positive for GLIS3, NKX2.1, and PAX8 signal, and the percent overlap between them are indicated. B Heatmap showing overlap between GLIS3, NKX2.1, and PAX8 binding. Heatmap of the 2 kb region centered on each of the binding regions with ChIP-seq signal normalized to 10 million reads. C Venn diagram showing the number of target genes with GLIS3, NKX2.1, and/or PAX8 binding regions. D Venn diagram showing the overlap of G⁺N⁺P⁺ genes with genes up- or down-regulated in the thyroid gland of Glis3KO-LID mice compared to that of WT-LID mice. E KEGG analysis of the 4502 G⁺N⁺P⁺ genes

Fig. 3

GLIS3, NKX2.1, and PAX8 share binding loci within the regulatory regions of several TH biosynthetic genes. A Colocalization of GLIS3, NKX2.1, and/or PAX8 ChIP-seq loci in genes critical for TH biosynthesis. The NUE region in Slc5a5 (Nis) is indicated. B, C NKX2.1 and PAX8 analysis at Cdh16 (− 0.2 kb), Slc5a5 (− 2.8 kb), and Tpo (− 0.1 kb) with thyroid glands from WT and Glis3KO mice. Binding to Gapdh and Tpo (− 1.9 kb) served as negative controls

Fig. 4

Genome browser tracks of several genes showing overlap of GLIS3, NKX2.1, and PAX8 binding loci in mouse thyroid gland. A Genes known to be induced by TSH. B Collagen and chemokine genes

Fig. 5

Global analysis of GLIS3, NKX2.1, PAX8, and FOXE1 genomic binding in rat thyroid follicular PCCl3 cells. A–D HOMER analysis, heatmap, and ChIP-Seq read density of GLIS3 (A), PAX8 (B), FOXE1 (C), and (D) NKX2.1 binding data. E Genomic context of the GLIS3, PAX8, FOXE1, and NKX2.1 peaks within the whole rat genome (rn6)

Fig. 6

GLIS3, NKX2.1, PAX8, and FOXE1 binding to the PCCl3 genome partially overlap. A Percent NKX2.1, PAX8, and FOXE1 binding loci overlapping with those of GLIS3. B Heatmaps of the 2 kb region centered on each of the binding regions with ChIP-seq signal normalized to 10 million reads shows overlaps between GLIS3, NKX2.1, PAX8, and FOXE1 binding. C Genome browser tracks of several thyroid genes showing overlap of GLIS3, NKX2.1, PAX8, and FOXE1 binding loci in PCCl3 cells

Fig. 7

Schematic view of GLIS3 regulation in the expression of target genes with other TFs. GLIS3 does not affect PAX8 or NKX2.1 binding nor the open/closed chromatin structure at their regulatory regions of several GLIS3 target genes but may promote transcriptional activation by enhancing the interaction of regulatory regions with Pol II complexes