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型同源盒基因，我们称之为Toto。Toto是一个不寻常的同源盒基因有两个原因。首先，它编码一个小蛋白质（73个氨基酸），几乎完全由一个同质结构域组成。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都是保守的，而且Toto不结合DNA。Toto编码一种8Kd核蛋白，在Nkx2.5表达后心肌细胞检测后不久表达。在Nkx2.5缺失胚中Toto基因明显下调，Nkx2.5在体外可直接激活Toto启动子。我们在小鼠中使用一种策略使Toto失活，从而导致LacZ在Toto表达域的表达。相当比例的Toto null胚胎在妊娠中期死亡，伴有心包积液和心肌层变薄，有时伴有心脏破裂和心包血肿。一些托托小鼠活到成年。初步数据表明，这些老鼠的心脏不正常。在培养细胞中，Toto负调控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。因此，Toto代表了一类新的同源结构域蛋白，它保留了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澄清Toto的功能：我们将通过破坏心肌蛋白、Nkx2.5和/或Gata4与SRF的关联，通过直接与心肌蛋白、Nkx2.5、Gata4和/或SRF相互作用，或通过阻止SRF与DNA相互作用，来确定Toto是否抑制Nkx2.5、Gata4和SRF依赖性基因转录的激活。2. 我们将在转基因小鼠发育中的心脏中过表达Toto，以确定Toto在体内过表达是否会下调心脏特异性基因的转录。3. 我们将确定Toto缺乏在正常情况下和肥厚刺激后成人心功能中的作用。

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.