Hox Genes & Lineage Infidelity

霍克斯基因

• 批准号: 10004644
• 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/10004644
• 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 简单生物体中的Hox基因突变常常导致一个基因的剧烈同源异型转化， 身体的一部分变成另一部分它们编码的转录因子可以启动基因级联反应， 细分市场的发展命运。在哺乳动物中，有39个Hox基因，分为四个簇，分为13个簇， 功能上相关的paradox。需要突变来自多个亲本群体的多个Hox基因 以克服冗余并揭示先前隐藏的共享功能。为此，我们制作了 在相邻的Hox基因组中有移码突变。通过杂交，我们可以降低Hox功能， 同时维持足够的Hox 11表达以具有发育中的肾脏进行研究。 具有12个密切相关的Hox 9、10、11等位基因的同时移码突变的小鼠显示出非常高的遗传学效应。 意外的肾脏表型。突变肾单位的细胞通常表现出混合特性， 表达一种以上节段细胞类型的标志物。这是一个广泛的电池检查， 片段特异性标记。进一步证实，果蝇中的Hox突变也可以导致去- 抑制了许多在其他谱系中正常表达的基因，与我们在小鼠中看到的非常相似。 在具体目标1中，我们建议进一步研究 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。有多少种不同的细胞类型标记物可以 是由单个细胞表达的吗突变体中的开/闭染色质构型是否揭示了更多 表观遗传可塑性比RNA-seq？是否存在暗示机制的通路扰动？ 在具体目标2中，我们检验了观察到的混合细胞同一性突变体表型是 Polycomb Repressive Complex（PRC）功能被破坏。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复合物识别并维持它们的表达。 镇压在果蝇中的工作表明，Hox蛋白和PRC蛋白可以共同结合，以驱动阻遏 不合适的细胞类型基因。为此，我们进行了一系列的Chip-seq实验，以比较野生型 PRC 1（H2 Aub 1）、PRC 2（H3 K27 me 3）、活性（H3 K4 me 3）、Hoxa 11和Hoxd 11的型/突变体分布 （使用我们的表位标记的小鼠），以及Ezh 2和Ring 1B PRC组分蛋白。 在具体目标3中，我们提出检验Hox基因也具有减少细胞类型的功能的假设。 成人的可塑性。Hox基因通常在成人中继续表达。为此， 仍然是不确定的，虽然有人提出，它有助于保持适当的表达， 分化基因我们认为，它也有助于维持对不适当分化的抑制 基因.我们建议对缺血再灌注野生型和Hox进行单细胞RNA-seq实验， 突变受损的肾脏，细胞通常去分化，然后重新分化，以验证他的假设。