Mechanisms of Pbx-directed Genetic &Transcriptional Control of Limb Development

Pbx定向遗传机制

• 批准号: 8238822
• 负责人: Licia Selleri
• 金额: $ 35.07万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-10 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238822
• 关键词: Ablation; Acute leukemia; Affect; Alleles; Beryllium; Binding; Binding Sites; Biochemical; Biological Phenomena; Body Patterning; Cell Culture Techniques; Cell Line; Cells; Chromatin; Comprehension; Congenital Abnormality; DNA Binding; Development; Digit structure; Distal; Drosophila genus; Electrophoretic Mobility Shift Assay; Elements; Embryo; Enhancers; Exhibits; Gene Expression; Gene Expression Regulation; Gene Family; Gene Targeting; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Transcription; Genitourinary system; Genomics; Homeobox Genes; Homeodomain Proteins; Homologous Gene; Human; In Vitro; Knowledge; Limb Bud; Limb Development; Limb structure; Live Birth; Mammals; Mesenchyme; Methods; Modeling; Molecular; Molecular Genetics; Morphogenesis; Mus; Neoplasms; Neuraxis; Nucleic Acid Regulatory Sequences; Nucleosomes; Organ; Organogenesis; Pathogenesis; Pattern; Play; Positioning Attribute; Proteins; Regulation; Regulatory Element; Research; Role; Skeletal Development; Skeleton; Solid Neoplasm; Spatial Distribution; Specificity; Staging; Structure; System; Testing; Tissues; Transcriptional Regulation; Transfection; Vertebrates; Work; appendage; base; chromatin immunoprecipitation; cofactor; fly; gastrointestinal; genetic regulatory protein; histone modification; in vivo; insight; leukemia; malformation; mouse model; mutant; novel; programs; research study; skeletal; transcription factor

DESCRIPTION (provided by applicant): In vertebrates, Hox genes play major roles in the formation of most vital organs. It has been proposed that the exquisite DNA-binding specificities that allow different Hox proteins to regulate specific target genes, thus instructing the identity of distinct body structures, depend on interactions with other homeoproteins, which act as Hox cofactors. For the last fifteen years, based on molecular and biochemical analyses, the prevailing view has been that TALE homeodomain proteins, which comprise the products encoded by the Pbx gene family, act as ancillary cofactors for Hox. Pbx1 is a homolog of Drosophila extradenticle (exd), which has critical roles in patterning of the fly body. While exd is the sole Pbx-encoding gene in the fly, the mouse has four such genes (Pbx1-4). Despite their paramount roles in organogenesis and patterning of the body and limb axes, the molecular mechanisms of Hox regulation remain elusive. Our objectives are to use the mouse limb as the most tractable and established system to delineate whether regulation of Hox "collinear" expression, a basic and mysterious biological phenomenon, is governed by Pbx. We have established that different Pbx genes, similarly to Hox genes, share overlapping roles in limb patterning and outgrowth. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit limb truncations similar to those of HoxA/D mutants. Additionally, we have found that Pbx1/Pbx2 control the onset and spatial distribution of 5' HoxA/D expression in limb mesenchyme. These findings establish that Pbx proteins hierarchically govern 5' HoxA/D gene expression in the limb. In view of these new findings, our hypothesis proposes a novel mechanism for Hox gene regulation, whereby 5' Hox expression is directly controlled at the transcriptional level by Pbx in the bud mesenchyme for limb morphogenesis and digit formation. We will test our hypothesis using embryologic, genetic and molecular approaches in the mouse. First, by molecular methods, we will determine whether Pbx1/2 regulate 5' HoxD transcription by direct control of the HoxD GCR, a genomic region that governs HoxD collinear expression in the autopod. We will then test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in the mouse. Moreover, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in the limb field and bud mesenchyme. By this approach, we will determine when Hox expression is first affected by Pbx loss in the limb bud. Completion of these studies will define novel regulatory networks that govern transcription of Hox genes and will directly contribute to the understanding of human congenital limb malformations. Broadly, given the involvement of human HOX genes in leukemias and solid tumors, our studies will inform general comprehension of HOX regulation also in human neoplasia. PUBLIC HEALTH RELEVANCE: Hox proteins play essential roles in the formation of many critical organs in mammals, including the limb; however, the molecular mechanisms underlying Hox gene regulation remain elusive. The proposed studies will use mouse models to provide novel insights into the control of Hox regulation by Pbx proteins in the limb. Accordingly, a broad impact of this work will be the generation of new knowledge on the pathogenesis of human congenital malformations, including those that affect limb skeletal development and function, occurring in approximately 1 of 500 human live births.

描述（由申请人提供）：在脊椎动物中，HOX基因在形成大多数重要器官的形成中起着重要作用。已经提出，允许不同HOX蛋白来调节特定靶基因的精美的DNA结合特异性，从而指导不同人体结构的身份，取决于与其他同源蛋白的相互作用，这些同源蛋白充当HOX辅助因子。在过去的十五年中，基于分子和生化分析，普遍的观点是，构成由PBX基因家族编码的产物的故事同源域蛋白充当HOX的辅助辅助因子。 PBX1是果蝇内部（EXD）的同源物，在蝇体模式中具有关键作用。虽然EXD是苍蝇中唯一的PBX编码基因，但小鼠具有四个这样的基因（PBX1-4）。尽管在身体和肢体轴的器官发生和模式中它们具有至关重要的作用，但HOX调节的分子机制仍然难以捉摸。我们的目标是将小鼠肢体用作最易加和既定的系统来描述HOX“ colinear”表达的调节（一种基本和神秘的生物学现象）是否由PBX控制。我们已经确定，与HOX基因相似的不同PBX基因在肢体图案和产物中具有重叠的角色。因此，PBX1/PBX2双重纯合（PBX1 - / - ; PBX2 - / - ）胚胎完全缺乏肢体，而PBX1 - / - ; PBX2突变体表现出类似于Hoxa/D突变体的肢体截断。此外，我们发现PBX1/PBX2控制肢体间充质中5'HOXA/D表达的发作和空间分布。这些发现表明，PBX蛋白在肢体中层次分层控制5'HOXA/D基因表达。鉴于这些新发现，我们的假设提出了一种新的HOX基因调节机制，从而通过PBX在芽中质中直接控制5'HOX表达在转录水平上，以实现肢体形态发生和数字形成。我们将使用小鼠中的胚胎学，遗传和分子方法检验我们的假设。首先，通过分子方法，我们将通过直接控制HOXD GCR来确定PBX1/2是否调节5'HOXD转录，这是一个控制自动脚架中HOXD共线表达的基因组区域。然后，我们将测试PBX与HOXD GCR的结合是否具有通过细胞培养中的瞬时转染和小鼠中瞬态转基因实验在转录方面的功能轴承。此外，通过组织特异性和可诱导的遗传消融，使用我们的新PBX1条件等位基因（在PBX2缺陷的背景上），以及可用的CRE系（其中一种在间质中诱导），我们将在Limb Field和BudeChememe的Limb Field和Budechmeememe中剖析PBX1/PBX1/PBX1/PBX2的空间和时间要求。通过这种方法，我们将确定何时首先在肢体芽中受PBX损失的影响。这些研究的完成将定义主管HOX基因转录的新型调控网络，并将直接有助于理解人类先天性肢体畸形。从广义上讲，鉴于人Hox基因参与白血病和实体瘤，我们的研究也将为人类肿瘤中的HOX调节提供一般理解。 公共卫生相关性：HOX蛋白在包括肢体在内的哺乳动物中的许多关键器官形成中起着重要作用；然而，HOX基因调节的分子机制仍然难以捉摸。拟议的研究将使用小鼠模型来提供新的见解，以控制肢体中PBX蛋白对HOX调节的控制。因此，这项工作的广泛影响将是关于人类先天性畸形的发病机理的新知识，包括影响肢体骨骼发育和功能的新知识，该知识发生在500个人类活产中约1个。