Thrsp/Spot14, thyroid hormone responsive protein; Cyp7a1, cytochrome P450 member 7a1; Slc2a2/Glut2, solute carrier family 2 member a2; G6pc, glucose-6-phosphatase catalytic subunit; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Ldlr, low density lipoprotein receptor; Acaca/ACC1, acetyl-CoA carboxylase 1; Cpt1a, carnitine palmitoyltransferase 1A; GSK3b, glycogen synthase kinase 3 ; Pdk2, pyruvate dehydrogenase kinase 2; THRB, thyroid hormone receptor ; K146, lysine 146; T3, triiodothyronine

Thrsp/Spot14, thyroid hormone responsive protein; Cyp7a1, cytochrome P450 member 7a1; Slc2a2/Glut2, solute carrier family 2 member a2; G6pc, glucose-6-phosphatase catalytic subunit; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Ldlr, low density lipoprotein receptor; Acaca/ACC1, acetyl-CoA carboxylase 1; Cpt1a, carnitine palmitoyltransferase 1A; GSK3b, glycogen synthase kinase 3 ; Pdk2, pyruvate dehydrogenase kinase 2; THRB, thyroid hormone receptor ; K146, lysine 146; T3, triiodothyronine. Thyroid status and changes in the pituitary and thyroid of THRB K146Q mice. Serum thyroxine (T4) and T3 levels in K146Q mice were not significantly different from control. the THRB K146Q mice was 10-fold lower (per gram tissue) than control, despite normal TSH bioactivity. The expression of thyroglobulin and dual oxidase 2 genes in the thyroid was reduced and associated with modifications of cAMP response elementCbinding protein DNA binding and cofactor interactions in the presence of the desumoylated THRB. Therefore, thyroid hormone production had both TSH-dependent and TSH-independent components. We conclude that THRB sumoylation at K146 was required for normal TSH feedback regulation and TH synthesis in the thyroid gland, by a TSH-independent pathway. and gene expression involves multiple transcription factors (TFs) in addition to THRB, including GATA-binding protein 2 (GATA2) and Pit 1/POU1F1 (6C12). The role of these TFs, however, has largely been based on transient reporter expression assays rather than direct determination of in vivo binding to the gene regulatory regions (11). Pit 1 is essential for normal anterior pituitary development, and individuals with a Pit 1 gene mutation in a region of the Pit 1 protein that disrupts function can develop anterior pituitary insufficiency, including central hypothyroidism and reduced growth hormone secretion (13). GATA2 is usually important for both stimulation and repression of gene expression (9, 14). GATA-mediated transcription regulation has 2 modules: friend of GATACdependent (FOG-dependent) and FOG-independent. FOG1 contains 8 zinc fingers and is the best characterized GATA factor. FOG1 heterodimerizes with GATA, recruits nucleosome remodeling factor and deacetylase, and then activates or represses transcription depending on the promoter context (15C18). FOG-dependent GATA action is involved in a range of diverse cellular processes (19, 20). We previously showed that THRA and beta-Eudesmol THRB were posttranslationally altered by small ubiquitin-like modifier (SUMO), important for TH/THR-mediated transcription (21). A number of TFs are sumoylated, including nuclear receptors, as well as kinases and phosphatases (22C25). SUMO modification involves the attachment of a SUMO, through covalently binding to a specific lysine motif in the target protein. beta-Eudesmol The modification is usually reversible by SUMO peptidases that break the bond, called desumoylation. Sumoylation and desumoylation dynamically influence proteinCprotein interactions, protein structural conformation, and protein binding to DNA. Sumoylation is usually involved in a range of biological processes, including DNA replication, RNA transcription, cell proliferation and differentiation, metabolism, immune response, and neuronal function (26C29). Sumoylation defects have Rabbit polyclonal to ESD been associated with several diseases. We previously reported that THRA has 2 sumoylation sites and THRB has 3 (21). Mutations at each of these sites reduce sumoylation in vitro and disrupt THR conversation with coactivators and corepressors. Reduction in THRA sumoylation was shown to impair Wnt signaling, resulting in reduced preadipocyte proliferation and interference with PPAR-mediated adipogenesis (21, 30). In vitro studies showed that this sumoylation of THRs is usually important for receptor function and TH signalCmediated cellular functions. To determine the physiological role of THR sumoylation in vivo, we generated a mouse carrying a mutation at lysine 146 (K146) in THRB that is required for SUMO conjugation. In this mouse model, we demonstrate that this sumoylation of THRB at K146 was essential for the regulation of gene expression and for normal TH production in the thyroid. Desumoylated THRB directly disrupted gene regulation, as well as disrupting cAMP response elementCbinding proteinCmediated (CREB-mediated) regulation of genes important for TH synthesis, including thyroglobulin (test in Prism statistical software. THRB, thyroid hormone receptor ; K146, lysine 146; DBD, DNA-binding domain name; LBD, ligand-binding domain name; Zn 2, second zinc finger; RXR, retinoid X receptor; DR4-TRE, direct repeat, 4-base pair gap, thyroid hormone response element. We generated a mouse model with a global THRB K146Q mutation, using traditional gene targeting techniques (Supplemental Physique 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.149425DS1). The mutation was generated by a single nucleoside substitution, changing AAG (K) to CAG (Q). The isoforms, “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001113417″,”term_id”:”164663876″,”term_text”:”NM_001113417″NM_001113417, “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_011244745″,”term_id”:”1907100331″,”term_text”:”XM_011244745″XM_011244745). The mutation position of nucleoside 1516 corresponds to the K146 residue in the THRB protein. The genotyping was based on the location in 0.04 is shown). Paired Students test with normal distribution was used in statistical analysis. The statistical analysis for data shown in all panels was performed using Students test for paired analysis. Thrsp/Spot14, thyroid hormone responsive protein; Cyp7a1, cytochrome P450 member 7a1; Slc2a2/Glut2, solute carrier beta-Eudesmol family 2 member a2; G6pc, glucose-6-phosphatase catalytic subunit; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Ldlr, low density lipoprotein receptor; Acaca/ACC1, acetyl-CoA carboxylase 1; Cpt1a, carnitine palmitoyltransferase 1A; GSK3b, glycogen synthase kinase 3 ; Pdk2, pyruvate dehydrogenase kinase 2; THRB, thyroid hormone receptor ; K146, lysine 146; T3, triiodothyronine. Thyroid status and changes in the pituitary and thyroid of THRB K146Q mice. Serum thyroxine (T4) and T3 levels in K146Q mice.