An unusual homeobox gene required for heart development

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盒基因，我们将其称为Toto。 Toto 是一种不寻常的同源框基因，原因有两个。首先，它编码一个几乎完全由同源域组成的小蛋白质（73 个氨基酸）。其次，它不包含在接触 DNA 的所有其他同源域中保守的某些氨基酸残基，并且 Toto 不结合 DNA。 Toto 编码 8Kd 核蛋白，并且在 Nkx2.5 表达后心肌细胞测定后不久表达。 Toto在Nkx2.5无效胚胎中显着下调，并且Nkx2.5可以在体外直接激活Toto启动子。我们使用导致 Toto 表达域中 LacZ 表达的策略灭活了小鼠中的 Toto。很大一部分 Toto 无效胚胎在妊娠中期因心包积液和心肌致密层变薄而死亡，有时与心脏破裂和心包血肿有关。一些托托缺失小鼠能活到成年。初步数据表明这些小鼠的心脏存在异常。在培养细胞中，Toto 负向调节心脏特异性转录途径，包括涉及 Nkx2.5、Gata4、SRF 和心肌素的转录途径。我们假设 Toto 通过直接与心脏特异性转录因子相互作用来负向调节 SRF 依赖性心脏转录发挥作用。因此，Toto代表了一类新的同源域蛋白，它保留了蛋白质-蛋白质相互作用的能力，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Gata4与SRF的关联，通过直接与心肌素、Nkx2.5、Gata4和/或SRF相互作用，或通过阻止SRF与DNA相互作用来抑制SRF依赖性基因转录的Nkx2.5、Gata4和心肌素激活。 2. 我们将在转基因小鼠发育中的心脏中过度表达Toto，以确定心脏特异性基因转录是否因体内Toto过度表达而下调。 3. 我们将确定正常条件下和肥大刺激后托托缺乏症对成人心脏功能的作用。

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.