Hox Genes & Lineage Infidelity

霍克斯基因

• 批准号: 10160899
• 负责人: S. Steven Potter
• 金额: $ 50.61万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10160899
• 关键词: ATAC-seq; Abies; Adult; Alleles; Automobile Driving; Binding; Biological Assay; Body part; Cell Differentiation process; Cells; ChIP-seq; Child; Chromatin; Code; Complex; Data; Data Set; Defect; Development; Differentiated Gene; Disease; Distal; Drosophila genus; Duct (organ) structure; Epigenetic Process; Epitopes; Frameshift Mutation; Gene Expression; Gene Mutation; Genes; Genetic; HOX protein; Head; Henle' s loop; Histones; Homeobox Genes; Immunofluorescence Immunologic; Individual; Injury to Kidney; Ischemia; Kidney; Lead; Lectin; Leg; Lysine; Mammals; Mediating; Modeling; Mus; Mutate; Mutation; Natural regeneration; Nephrons; Organ; Organism; PRC1 Protein; Paper; Pathway interactions; Phenotype; Polycomb; Prevention; Proteins; Regulator Genes; Renal tubule structure; Reperfusion Therapy; Repression; Research; Role; Scientist; Series; Stains; Testing; Tissues; Ubiquitination; Uncertainty; Work; base; cell type; combinatorial; experimental study; gene function; gene repression; imaginal disc; insight; kidney cell; mutant; novel; paralogous gene; preservation; protein complex; repaired; single-cell RNA sequencing; transcription factor; transcriptome sequencing

Abstract Hox gene mutations in simpler organisms often resulting in dramatic homeotic transformations of one body part into another. They encode transcription factors that can initiate genetic cascades that drive the developmental destinies of segments. In mammals there are 39 Hox genes, in four clusters, divided into 13 functionally related paralog groups. It is necessary to mutate multiple Hox genes from multiple paralog groups to overcome redundancies and reveal previously hidden shared functions. To this end we have made mice with frameshift mutations in sets of adjacent Hox genes. By interbreeding we can dial down Hox function for multiple paralog groups while maintaining sufficient Hox11 expression to have a developing kidney to study. Mice with simultaneous frameshift mutation of twelve closely related Hox9,10,11 alleles show a very unexpected kidney phenotype. The cells of the mutant nephrons often show mixed identities, with co- expression of markers of more than one segment cell type. This was examined with an extensive battery of segment specific markers. Providing further confirmation, Hox mutation in Drosophila can also lead to de- repression of many genes normally expressed in other lineages, very similar to what we see in mice. In specific aim 1 we propose to further study the apparently confused, mixed identity character of the multi-Hox mutants using single cell RNA-seq and single cell ATAC-seq, to define the limits of the crossing of lineage boundaries and to search for underlying mechanisms. How many different cell type markers can be expressed by an individual cell? Do open/closed chromatin configurations in mutants reveal even more epigenetic plasticity than seen by RNA-seq? Are there perturbations in pathways that suggest mechanisms? In specific aim 2 we test the hypothesis that the observed mixed cell identity mutant phenotype is the result of disrupted Polycomb Repressive Complex (PRC) function. Cell type specific transcription factors initially establish repressed gene expression states, and then PRC complexes recognize and maintain their repression. Work in Drosophila has shown that Hox proteins and PRC proteins can co-bind to drive repression of inappropriate cell type genes. In this aim we carry out a series of Chip-seq experiments to compare wild type/mutant distributions of PRC1 (H2Aub1), PRC2 (H3K27me3), active (H3K4me3), Hoxa11 and Hoxd11 (using our epitope tagged mice), as well as Ezh2 and Ring1B PRC component proteins. In specific aim 3 we propose to test the hypothesis that Hox genes also function to reduce cell type plasticity in the adult. Hox genes generally continue to be expressed in the adult. The function for this has remained uncertain, although it has been proposed that it serves to maintain expression of the appropriate differentiation genes. We propose that it also serves to maintain repression of inappropriate differentiation genes. We propose to carry out single cell RNA-seq experiments on ischemia reperfusion wild type and Hox mutant injured kidneys, where cells normally de-differentiate and then re-differentiate, to test his hypothesis.

摘要 在更简单的生物体中，HOX基因突变经常导致一种戏剧性的同源异型转变 身体的一部分变成另一个。它们编码的转录因子可以启动遗传级联反应，从而推动 区段的发展命运。在哺乳动物中，有39个HOX基因，分成4个簇，分为13个 功能相关的Paralog组。从多个平行对数组中突变多个HOX基因是必要的 以克服冗余并揭示以前隐藏的共享功能。为此，我们制造了老鼠 一组相邻的HOX基因发生移码突变。通过杂交，我们可以降低HOX的功能 多个Paralog组，同时保持足够的Hox11表达，以有一个发育中的肾脏进行研究。 12个密切相关的Hox9，10，11等位基因同时发生移码突变的小鼠表现出非常 意外的肾脏表型。突变的肾单位的细胞通常表现出混合的身份，与共同的- 表达一种以上节段细胞类型的标记。对此进行了广泛的研究， 特定于段的标记。进一步证实，果蝇体内的HOX突变也可以导致去甲基化。 抑制许多基因在其他谱系中正常表达，与我们在老鼠身上看到的非常相似。 在具体目标1中，我们建议进一步研究明显混乱的、混合的身份特征 使用单细胞RNA-seq和单细胞atac-seq的多Hox突变体，以定义 血统界限和寻找潜在的机制。可以有多少种不同的细胞类型标记 是由单个细胞表达的？突变体的开放/闭合染色质配置是否揭示了更多 表观遗传可塑性比RNA-Seq？在暗示机制的通路中是否存在扰动？ 在特定目标2中，我们检验了这样的假设，即观察到的混合细胞身份突变表型是 多梳抑制复合体(PRC)功能中断的结果。细胞类型特异性转录因子 最初建立被抑制的基因表达状态，然后PRC复合体识别并维持其 压抑。对果蝇的研究表明，Hox蛋白和PRC蛋白可以共同结合来驱动抑制 不适当的细胞类型基因。为了达到这个目的，我们进行了一系列的芯片序列实验，以比较野生型 Prc1(H_2Aub1)、PrC2(H3K27me3)、Active(H3K4me3)、Hoxa11和Hoxd11的类型/突变分布 (使用我们的表位标记的小鼠)，以及Ezh2和Ring1b PRC组件蛋白。 在特定的目标3中，我们建议检验HOX基因也能减少细胞类型的假设 成人的可塑性。HOX基因通常在成人体内继续表达。此功能的函数有 仍然不确定，尽管有人提议，这有助于维持适当的 分化基因。我们认为，这也有助于保持对不适当分化的压制。 基因。我们建议开展缺血再灌注野生型和HOX单细胞rna-seq实验。 突变的受损肾脏，在那里细胞通常去分化，然后再分化，以检验他的假设。