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个在心脏发育过程中表达的新的同源框基因，我们称之为TOTO。Toto是一种不同寻常的同源盒基因，原因有两个。首先，它编码一种小蛋白质(73个氨基酸)，几乎完全由同源蛋白组成。其次，它不包含某些氨基酸残基，这些氨基酸残基在所有其他与DNA接触的同源结构域中是保守的，TOTO不与DNA结合。TOTO编码一个8Kd的核蛋白，在NKX2.5表达后，在心肌细胞测定后不久表达。在NKX2.5缺失的胚胎中Toto的表达显著下调，NKX2.5在体外可以直接激活Toto启动子。我们已经在小鼠中灭活了Toto，使用了一种策略，导致了Toto表达结构域中LacZ的表达。相当大比例的Toto零胚胎在妊娠中期死亡，伴有心包积液和心肌致密层变薄，有时与心脏破裂和心包血肿有关。一些Toto基因缺失的小鼠可以活到成年。初步数据显示，这些老鼠的心脏不正常。在培养的细胞中，Toto负向调节心脏特异转录途径，包括涉及NKX2.5、Gata4、SRF和myocardin的那些途径。我们假设Toto通过直接与心脏特异转录因子相互作用负向调节依赖SRF的心脏转录来发挥作用。因此，Toto代表了一类新的同源结构域蛋白，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Myocardin、NKX2.5和/或Gata4与SRF的关联、通过直接与Myocardin、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依赖的基因转录的NKX2.5、Gata4和myocardin的激活。2.我们将在转基因小鼠的发育心脏中过表达Toto，以确定在体内过表达Toto是否下调了心脏特异基因的转录。3.研究正常情况下和肥大刺激后Toto缺乏症对成人心功能的影响。

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.