A role for Hox-dependent chromatin architecture in defining cell-fate

描述(由申请人提供)：HOX基因家族编码一组同源结构域，包含所有后生动物发育所必需的转录调节因子。在发育过程中，HOX基因沿前后轴的表达对于确定细胞和组织同一性的遗传程序是必要的；然而，HOX基因的错误表达和错误调控与肿瘤的发生密切相关。然而，对HOX依赖的转录调控的分子机制缺乏完整的了解，阻碍了我们设计针对这些因素的癌症治疗药物的能力。这一建议的长期目标是通过具体解决以下假设来更好地理解这些机制：Hox转录调控因子通过特定的基因组结构定义细胞命运，导致广泛的基因表达变化。由于基因组结构的改变也与肿瘤发生相关，这一提议有可能提供对多水平过程的洞察，当改变时，具有启动和维持促进癌细胞形成的异常基因表达程序的能力。为了验证这一假说，这里提出的研究将集中在黑腹果蝇的翅膀和龟头的发育上，它们来自一系列同源的幼虫成像盘，只是在单个HOX基因的表达上存在差异，超双胸(UBX)。在龟头中表达UBX是必要的，也是改变遗传的充分条件。 计划，否则将导致翅膀的形成，因为Ubx的突变导致四翼苍蝇。在赞助商实验室开发的一项名为细胞和基因特异性染色质免疫沉淀(CgChIP)的新技术的基础上，我将分析与遗传层次结构的多个水平上的基因相关的调节区的3D基因组结构，这些基因促进了对Ubx的反应形成卤化物。具体地说，我会问，在Ubx基因座上维持Ubx表达的区域和在Ubx靶基因上维持Ubx表达的区域，是否存在基因组结构上的差异。在确定这些基因座存在差异后，我将确定Ubx的表达和活性是否对于建立在龟头中发现的基因组结构是必要的，从而提供对Ubx和其他Hox基因如何定义细胞命运的机械性洞察。后生动物中HOX基因的进化保守使得从果蝇的这项工作中获得的基本见解可以转化为理解高等生物中依赖于HOX的基因组结构规范，并最终发现癌症治疗干预的新靶点。

DESCRIPTION (provided by applicant): The family of Hox genes encodes a set of homeodomain containing transcriptional regulators that are necessary for the development of all metazoans. Expression of Hox genes along the antero-posterior axis is necessary to set the genetic program that defines cell- and tissue-identity during development; however misexpression and misregulation of Hox genes has been strongly linked to oncogenesis. The absence of a complete understanding of the molecular mechanisms of Hox-dependent transcriptional regulation, though, hampers our ability to design cancer therapeutics targeted against these factors. The long term objective of this proposal is to better understand these mechanisms by specifically addressing the hypothesis that Hox transcriptional regulators define cell-fate through a specification of genome architecture, resulting in wide-spread gene expression changes. As alterations in genome architecture have also been correlated with oncogenesis, this proposal has the potential to provide insight into the multi-level process that, when altered, has the capacity to initiate and maintain the abnormal gene expression programs that promote cancer-cell formation. To test this hypothesis, the studies proposed here will focus on the development of the wing and haltere in Drosophila melanogaster, which are derived from serially homologous larval imaginal discs that differ only in the expression of a single Hox gene, Ultrabithorax (Ubx). Ubx expression in the haltere is necessary and sufficient to alter the genetic program that would otherwise result in the formation of the wing, as mutations in Ubx result in four-winged flies. Expanding upon a novel technology developed in the sponsor's laboratory, termed Cell- and Gene-specific Chromatin Immunoprecipitation (cgChIP), I will assay the 3D genomic architecture of regulatory regions associated with genes at many levels of the genetic hierarchy that promote haltere formation in response to Ubx. Specifically, I will ask whether any differences in genomic architecture exist in the wing versus the haltere at regions in the Ubx locus that function to maintain Ubx expression in the ON/OFF state and at Ubx-target genes. Having established that differences exist at these loci, I will determine if Ubx expression and activity is necessary to establish the genome architecture found in the haltere, thus providing mechanistic insight into how Ubx, and other Hox genes, define cell-fate. The evolutionary conservation of Hox genes throughout metazoans allows for the fundamental insights gained from this work in Drosophila to be translated into an understanding of Hox-dependent specification of genome architecture in higher organisms, and ultimately into the discovery of novel targets for cancer therapeutic intervention.