Thyroid Hormone Nuclear Receptors in Health and Disease

健康和疾病中的甲状腺激素核受体

• 批准号: 10262028
• 负责人: SHEUE-YANN CHENG
• 金额: $ 86.92万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262028
• 关键词: Adolescent; Adult; Affect; Amino Acid Sequence Homology; Anemia; Attention deficit hyperactivity disorder; Binding; Biological Process; Bone Development; Bone Marrow; C-terminal; CRISPR/Cas technology; Cardiac; Constipation; DNA; DNA Sequence; Defect; Development; Disease; Dominant-Negative Mutation; Embryo; Embryonic Development; Erythrocytes; Erythroid; Erythropoiesis; Exhibits; Fertility; Fishes; Gene Duplication; Gene Expression; Gene Expression Regulation; Genes; Growth; Growth and Development function; Health; Hormones; Human; Hypothyroidism; Impairment; Insulin-Like Growth Factor I; Intestinal Diseases; Intestines; Keratin; Larva; Lead; Ligands; Mediating; Messenger RNA; Modeling; Molecular; Morphology; Mus; Muscle; Mutagenesis; Mutant Strains Mice; Mutation; Nuclear Hormone Receptors; Pathologic; Patients; Phenotype; Pituitary Gland; Protein Isoforms; Proteins; Repression; Research; Resistance; Role; Serum; Somatotropin; Symptoms; THRA gene; THRB gene; Therapeutic; Thyroid Hormone Receptor; Thyroid Hormone Receptor Gene; Thyroid Hormones; Thyrotropin; Tissues; Villus; Visualization; Zebrafish; genomic locus; hearing impairment; hormone response element; human disease; in vivo; intestinal crypt; keratinocyte; mouse model; mutant; novel; progenitor; promoter; receptor; screening; skin organogenesis; stem cell proliferation; transcription factor; transcriptome sequencing

Understanding of the in vivo molecular actions of the TRalpha1 mutant has been limited since the initial identification of patients with THRA mutations. However, by using Thra1PV/+ mice we have elucidated how TRalpha1 mutants act to cause pathological manifestations in patients, such as erythroid and intestine disorders. We found that TRalpha1PV acts to suppress the clonogenic potential of progenitors in the erythrocytic lineage in bone marrow, leading to a reduction of mature erythrocytes. Further, we found that a key driver in the erythropoiesis, the Gata-1 gene, is a T3-directly regulated gene. TRalpha1PV impairs erythropoiesis, at least in part, via repression of the Gata-1 gene expression and its downstream regulated genes, causing anemia. We also demonstrated that adult Thra1PV/+ mice exhibit constipation as do RTHalpha patients. Further, we detected shorter villi and reduced stem-cell proliferation in the intestine crypts of Thra1PV/+ mice. These findings further demonstrated that we can use the Thra1PV/+ mouse to analyze in depth the pathogenetic actions of TRalpha1 mutants in human RTHalpha. Recently we have generated novel RTHalpha models in zebrafish to facilitate the study of the pathogenetic actions of TRalpha1 mutants in early development. We cannot use the Thra1PV/+ mouse to study TRalpha1 mutants in early development because of the insurmountable difficulty obtaining sufficient mutant embryos. Zebrafish have been increasingly used as models for human diseases owing to their high fecundity, rapid external embryonic development, and the easy visualization of transparent embryos. The zebrafish have two duplicated thra genes, thraa and thrab. Their encoded receptors show a high extent of amino acid sequence homology in the functional DNA and hormone binding domains with those of human and mouse TRalpha1 (90-95%). Using CRISPR/Cas9-mediated targeted mutagenesis, we created germline transmittable mutant fish. The stable expression of thra mutants from embryos to adulthood facilitated the study of molecular actions of TRalpha1 mutants during embryonic development as well as post-embryonic development. Two mutant fish lines expressing a mutation in the thraa gene (thraa 8-bp insertion mutant gene, encoding ThraaLeu405Glufs*6 mutant receptor) or the thrab gene (thrab 1-bp insertion mutant gene, encoding ThrabGlu394* mutant receptor) were obtained. These C-terminal truncation dominant negative mutant receptors were similar to those identified in patients. Adult and juvenile ThrabGlu394* mutants exhibited severe growth retardation, but adult ThraaLeu405Glufs*6 mutants had only very mild growth impairment. Expression of growth hormone in the pituitary and insulin-like growth factor 1 in muscle was markedly suppressed in ThrabGlu394* mutants. Decreased mRNA and protein levels of T3-regulated keratin genes and inhibited keratinocyte proliferation resulted in epidermal hypoplasia in adult and juvenile ThrabGlu394* mutants, but not ThraaLeu405Glufs*6 mutant fish. RNA-seq analysis showed that ThrabGlu394* mutation had global impact on the functions of the adult pituitary. However, neither morphological defects nor any changes in the expression of gh1 and keratin genes were observed in the embryos and early larvae. Thus, mutations of either the thraa or thrab gene did not affect initiation of embryogenesis. Rather, the mutation of the thrab gene, but not the thraa gene, is detrimental in post-larva growth and skin development. These differential effects suggested that the thra duplicated genes are essential to control temporal coordination in post-larva growth and development in a tissue-specific manner. Our studies uncovered novel functions of the duplicated thra genes in zebrafish in development. These mutant zebrafish could be used as models for further analysis of in vivo actions of TRalpha1 mutants during development and for large-scale screening of therapeutics for RTHalpha.

自从最初鉴定出患有THRA突变的患者以来，对Tralpha1突变体的体内分子作用的理解一直受到限制。但是，通过使用thra1pv/+小鼠，我们阐明了tralpha1突变体的作用如何引起患者（例如红细胞和肠道疾病）的病理表现。我们发现tralpha1pV起着抑制骨髓红细胞谱系中祖细胞的克隆生成潜力，从而导致成熟的红细胞减少。此外，我们发现红细胞生成的关键驱动器，GATA-1基因是T3直接调节的基因。 tralpha1pv至少部分通过抑制GATA-1基因表达及其下游调节基因，从而损害了红细胞生成，从而导致贫血。我们还证明了成年的Thra1pv/+小鼠与Rthalpha患者一样表现出便秘。此外，我们在Thra1pv/+小鼠的肠道隐窝中检测到较短的绒毛和减少的干细胞增殖。这些发现进一步表明，我们可以使用thra1pv/+小鼠在人类rthalpha中tralpha1突变体的致病作用进行深入分析。最近，我们在斑马鱼中生成了新型的Rthalpha模型，以促进对早期发育中Tralpha1突变体的致病作用的研究。由于无法获得足够的突变胚胎，我们无法使用Thra1pv/+小鼠在早期发育中研究Tralpha1突变体。斑马鱼越来越多地用作人类疾病的模型，因为它们的高繁殖力，快速的外部胚胎发育以及透明胚胎的易于可视化。斑马鱼具有两个重复的Thra基因，Thraa和Thrab。它们的编码受体显示在功能性DNA和激素结合结构域中与人和小鼠Tralpha1（90-95％）的氨基酸序列同源性。使用CRISPR/CAS9介导的靶向诱变，我们创建了可传输的种系。 Thra突变体从胚胎到成年的稳定表达促进了胚胎发育过程中Tralpha1突变体的分子作用以及胚胎后发育的研究。在ThraA基因中表达突变的两种突变鱼类系（ThraA 8-BP插入突变基因，编码Thraaleu405Glufs* 6突变受体）或THRAB基因（THRAB 1-BP插入突变体基因，编码Thrabglu394*突变受体）。这些C末端截断主要的负突变受体与患者鉴定的受体相似。成人和少年毛刺394*突变体表现出严重的生长迟缓，但成年thraaleu405glufs* 6个突变体仅具有非常轻微的生长障碍。垂体和胰岛素样生长因子1中生长激素在肌肉中的表达明显抑制。 T3调节的角蛋白基因的mRNA和蛋白质水平降低并抑制角质形成细胞增殖，导致成人和少年thrabglu394*突变体的表皮发育不全，但不是thraaleu405glufs* 6突变剂。 RNA-seq分析表明，Thabglu394*突变对成人垂体的功能具有全球影响。然而，在胚胎和早期幼虫中均未观察到GH1和角蛋白基因表达的形态缺陷或任何变化。因此，ThraA或Thrab基因的突变不会影响胚胎发生的开始。相反，THAR基因的突变，而不是ThraA基因，对雷尔瓦后的生长和皮肤发育有害。这些差异效应表明，THRA重复的基因对于以组织特异性方式控制了在多拉瓦后生长和发育中的时间协调至关重要。我们的研究发现了斑马鱼在发育中重复的THRA基因的新功能。这些突变体斑马鱼可以用作模型，以进一步分析发育过程中tralpha1突变体的体内作用和对rthalpha治疗的大规模筛查。