Defining the thyroid hormone receptor Cistrome in vivo

体内甲状腺激素受体 Cistrome 的定义

• 批准号: 8299630
• 负责人: ANTHONY N HOLLENBERG
• 金额: $ 21.75万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-11 至 2013-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299630
• 关键词: Adenoviruses; Adult; American; Antibodies; Area; Binding; Binding Sites; Biotin; Biotinylation; Birth; Body Weight; Cell Line; Cells; Childhood; Chromatin; Clinical; DNA; DNA Binding; Development; Energy Metabolism; Ensure; Family; Future; Gene Expression; Gene Targeting; Genes; Genomics; Grant; Growth; Growth and Development function; Health; Heart Diseases; Human; Human Development; Hyperthyroidism; Hypothyroidism; In Vitro; Knock-in Mouse; Knockout Mice; Learning; Life; Ligands; Ligase; Liver; Mediating; Metabolism; Modeling; Molecular; Mus; Neonatal; Neurologic; Newborn Infant; Nuclear; Nuclear Receptors; Nucleic Acid Regulatory Sequences; Pathway interactions; Peptides; Play; Positioning Attribute; Process; Promoter Regions; Protein Isoforms; Proteins; Regulation; Regulatory Element; Response Elements; Role; Signal Transduction; Specificity; Streptavidin; System; Technology; Testing; Thyroid Hormone Receptor; Thyroid Hormones; Thyroxine; Tissues; Work; base; chromatin immunoprecipitation; design; hormone response element; human disease; in vivo; innovation; insight; lipid metabolism; mRNA Expression; mouse model; novel; prevent; receptor; receptor binding; receptor coupling; response

DESCRIPTION (provided by applicant): Thyroid hormone (TH) is critical for normal development in murine models and humans. In addition, TH is a critical regulator of metabolism, growth and body weight in both childhood and adult life. TH mediates its effects by signaling through its cognate nuclear receptors, the thyroid hormone receptor isoforms (TRs). The TRs, through their interactions with coregulators proteins that vary depending upon the presence of TH, regulate gene expression positively or negatively. Based on significant work in vitro and in cell lines, it has been presumed that the effects of the TR on gene expression are dictated by the presence of TR response elements (TREs) in the regulatory regions of targeted genes. However, this has never been shown in vivo in key target tissues of TH action such as the liver, because current antibodies directed against the TR coupled with the amount of expressed TR in target tissues has made in vivo chromatin imunoprecipitation (ChIP) very difficult. To attack this issue and define TR binding sites in vivo, we will develop a peptide-tagged TR21 isoform that can be biotinylated in vivo by the biotin ligase BirA. The biotinylated TR21 isoform can then be precipitated in ChIP by streptavidin and the recovered DNA subjected to deep parallel sequencing (ChIP-seq) to define the TR cistrome in target issues. In the first aim of this proposal the tagged-TR21 isoform will be delivered to livers of TR2 knockout mice that express BirA by adenovirus followed by ChIP-seq to define the TR cistrome in liver. In the second aim, the biotinylation peptide will be knocked in to the endogenous TR21 genomic locus to allow for the development of a mouse model where ChIP-seq can be performed in any tissue as long as BirA is expressed. Together, completion of these Aims will provide a new platform for understanding TR action in vivo and allow for greater insight into how TH regulates key transcriptional pathways in vivo.

描述（由申请人提供）：甲状腺激素（TH）对于小鼠模型和人类的正常发育至关重要。此外，TH 是儿童期和成年期新陈代谢、生长和体重的关键调节剂。 TH 通过其同源核受体（甲状腺激素受体亚型 (TR)）发出信号来介导其作用。 TR 通过与根据 TH 的存在而变化的共调节蛋白的相互作用，正向或负向调节基因表达。基于体外和细胞系中的重要工作，推测 TR 对基因表达的影响是由靶基因调控区域中 TR 反应元件 (TRE) 的存在决定的。然而，这一点从未在体内 TH 作用的关键靶组织（如肝脏）中得到证实，因为目前针对 TR 的抗体与靶组织中表达的 TR 的量相结合，使得体内染色质免疫沉淀 (ChIP) 非常困难。为了解决这个问题并定义体内 TR 结合位点，我们将开发一种肽标记的 TR21 同工型，可以通过生物素连接酶 BirA 在体内进行生物素化。然后可以通过链霉亲和素在 ChIP 中沉淀生物素化的 TR21 同工型，并对回收的 DNA 进行深度平行测序 (ChIP-seq)，以定义目标问题中的 TR 顺反子。在该提案的第一个目标中，将通过腺病毒将标记的 TR21 同种型递送至表达 BirA 的 TR2 敲除小鼠的肝脏，然后进行 ChIP-seq 来定义肝脏中的 TR 顺反子。第二个目标是，将生物素化肽敲入内源 TR21 基因组位点，以开发小鼠模型，只要 BirA 表达，就可以在任何组织中进行 ChIP-seq。总之，这些目标的完成将为理解 TR 体内作用提供一个新平台，并允许更深入地了解 TH 如何调节体内关键转录途径。