An unusual homeobox gene required for heart development

心脏发育所需的不寻常同源盒基因

• 批准号: 6767694
• 负责人: Jonathan A. Epstein
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767694
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; cardiogenesis; electrocardiography; functional /structural genomics; gel mobility shift assay; gene expression; gene interaction; genetic regulation; genetic transcription; genetically modified animals; green fluorescent proteins; heart function; homeobox genes; immunoprecipitation; laboratory mouse; magnetic resonance imaging; nuclear proteins; posttranslational modifications; protein protein interaction; protein structure function; southern blotting; telemetry; transcription factor; western blottings

DESCRIPTION (provided by applicant): We have recently identified and cloned a novel homeobox gene expressed throughout cardiac development that we have called Toto. Toto is an unusual homeobox gene for two reasons. First, it encodes a small protein (73 amino acids) that is composed almost entirely of a homoedomain. Second, it does not contain certain amino acid residues conserved amongst all other homeodomains that contact DNA, and Toto does not bind DNA. Toto encodes an 8Kd nuclear protein and it is expressed shortly after cardiac myocyte determination following expression of Nkx2.5. Toto is markedly down-regulated in Nkx2.5 null embryos, and Nkx2.5 can directly activate the Toto promoter in vitro. We have inactivated Toto in the mouse using a strategy that results in the expression of LacZ in the Toto expression domain. A significant proportion of Toto null embryos die during mid gestation with pericardial effusions and a thinned compact layer of the myocardium, sometimes associated with cardiac rupture and pericardial hematoma. Some Toto null mice live to adulthood. Preliminary data suggests that these mice have abnormal hearts. In cultured cells, Toto negatively regulates cardiac specific transcriptional pathways including those that involve Nkx2.5, Gata4, SRF and myocardin. We hypothesize that Toto functions by directly interacting with cardiac specific transcription factors to negatively regulate SRF-dependent cardiac transcription. Thus, Toto represents a new class of homeodomain proteins that has retained protein-protein interaction capabilities, but has lost sequence specific DNA binding capacity. We will clarify Toto function by addressing the following aims: 1. We will determine whether Toto inhibits Nkx2.5, Gata4 and myocardin activation of SRF-dependent gene trancription by disrupting the association of myocardin, Nkx2.5 and/or Gata4 with SRF, by interacting directly with myocardin, Nkx2.5, Gata4 and/or SRF, or by preventing SRF from interacting with DNA. 2. We will over-express Toto in the developing heart of transgenic mice to determine if cardiac-specific gene transcription is down-regulated by Toto over-expression in vivo. 3. We will determine the role of Toto deficiency in adult cardiac function under normal conditions and after hypertrophic stimuli.

描述（由申请人提供）：我们最近鉴定并克隆了一种在心脏发育过程中表达的新型同源异型盒基因，我们称之为Toto。托托是一个不寻常的同源异型盒基因，原因有二。首先，它编码一种几乎完全由同源内切酶组成的小蛋白（73个氨基酸）。第二，它不包含在所有其他接触DNA的同源结构域中保守的某些氨基酸残基，并且Toto不结合DNA。Toto编码一个8 Kd的核蛋白，在Nkx2.5表达后心肌细胞确定后不久表达。Toto在Nkx2.5缺失的胚胎中表达显著下调，Nkx2.5在体外可直接激活Toto启动子。我们使用导致LacZ在Toto表达结构域中表达的策略在小鼠中灭活了Toto。很大一部分Toto null胚胎在妊娠中期死亡，伴有心包积液和心肌致密层变薄，有时伴有心脏破裂和心包血肿。一些Toto null小鼠活到成年。初步数据表明，这些老鼠有异常的心脏。在培养的细胞中，Toto负调节心脏特异性转录途径，包括涉及Nkx2.5，Gata 4，SRF和myocardin的那些。我们推测Toto通过直接与心脏特异性转录因子相互作用来负调节SRF依赖的心脏转录。因此，Toto代表了一类新的同源结构域蛋白，其保留了蛋白质-蛋白质相互作用能力，但失去了序列特异性DNA结合能力。我们将通过解决以下目标来阐明Toto的功能：1.我们将确定Toto是否通过破坏心肌素、Nkx2.5和/或Gata 4与SRF的结合，通过与心肌素、Nkx2.5、Gata 4和/或SRF直接相互作用，或通过阻止SRF与DNA相互作用来抑制Nkx2.5、Gata 4和心肌素对SRF依赖性基因转录的激活。2.我们将在转基因小鼠的心脏发育中过表达Toto，以确定心脏特异性基因转录是否被体内Toto过表达下调。3.我们将确定在正常条件下和肥大刺激后，Toto缺乏症在成人心脏功能中的作用。