Plasticity in an embryonic gene regulatory network

摘要 这项拟议研究的主要目的是阐明遗传和疾病的分子基础。 一个主要发育基因调控网络(GRN)的表观遗传变异。来自该实验室和其他实验室的研究 已经确定了一系列合子表达的核心GATA型转录因子，以及母体 调控输入，激活控制线虫内胚层发育的GRN。后者 包括母体提供的SKN-1/Nrf2转录因子和三重冗余的Wnt、MAPK和src 通过LIT-1/NLK激酶和POP-1/Tcf/Lef转录因子启动的信号系统 内胚层发育。移除这些输入中的任何一个都会导致不可渗透的表型，反映出 双稳状态，在遗传上不同的同种类型之间表现出广泛的差异。正反交组合的分析 在这一过程中，同型之间对SKN-1的定量要求不同，揭示了内胚层 GRN的产量也受到自然线虫之间不同的长期可遗传表观遗传状态的影响 同种类型。这种跨代表观遗传(TEI)需要参与piRNA功能的基因，即 核RNAi途径和组蛋白H3K9甲基化。这些发现为揭示 调节内胚层GRN的遗传和表观遗传可塑性的分子基础。在目标1中，我们将 评估关于三个基因不同地改变需求的作用机制的假设 用于SKN-1和WNT信令。我们将评估内胚层发育的核心调控因子如何表达 受母体GRN投入需求数量变化的影响。我们将评估如何 对LIT-1激酶需求的变化适应于控制不对称细胞的机制 分裂导致内胚层GRN的激活，并将检验以下假设： 对LIT-1的需求延伸到它在许多不对称细胞分裂中的全局作用。在目标2中，我们将开发 并实施高分辨率、高通量的方法来识别潜在的 对主要内胚层调节输入的要求。我们将测试候选基因的调节作用。 SKN-1依赖的内胚层GRN的激活。在目标3中，我们将分析分子基础 GRN输出的跨代遗传(TEI)。我们将评估内胚层GRN中的各个阶段 并检验SKN-1需求的表观遗传差异在 选定的同种类型延伸到其他调节输入。我们将检验TEI是由差异产生的假设 内胚层基因染色质状态及非编码RNA和内胚层差异表达 调控基因与TEI相关。这项研究的发现将有助于阐明生育的机制。 并且可以提供一个范例来理解个体的基因和基因之间的关系 对药理药物的反应，对推进精准医学具有重要意义。他们还将揭晓 改变Wnt信号转导结果的因素，Wnt信号转导是与许多癌症相关的主要调控机制。

SUMMARY The major objective of the proposed research is to illuminate the molecular basis underlying genetic and epigenetic variation in a major developmental gene regulatory network (GRN). Studies from this and other labs have identified a cascade of “core” zygotically expressed GATA-type transcription factors, and maternal regulatory inputs, that activate the GRN controlling development of the endoderm in C. elegans. The latter include the maternally supplied SKN-1/Nrf2 transcription factor and a triply redundant Wnt, MAPK, and src signaling system that acts through the LIT-1/NLK kinase and the POP-1/Tcf/Lef transcription factor to initiate endoderm development. Removal of any one of these inputs results in an impenetrant phenotype, reflecting a bistable state that shows wide variation between genetically distinct isotypes. Analysis of reciprocal crosses between isotypes with quantitatively different requirements for SKN-1 in this process revealed that endoderm GRN output is also influenced by long-term heritable epigenetic states that differ between natural C. elegans isotypes. This transgenerational epigenetic inheritance (TEI) requires genes involved in piRNA function, the nuclear RNAi pathway, and histone H3K9 methylation. These findings provide a springboard for unveiling the molecular basis for genetic and epigenetic plasticity in the regulation of the endoderm GRN. In Aim 1, we will evaluate hypotheses regarding the mechanisms of action of three genes that differentially alter the requirements for SKN-1 and Wnt signaling. We will assess how expression of the core regulators of endoderm development is influenced by quantitative variation in the requirement for the maternal GRN inputs. We will assess how variation in the requirement for LIT-1 kinase is accommodated in the mechanism that controls asymmetric cell division leading to activation of the endoderm GRN and will test the hypothesis that quantitative variation in the requirement for LIT-1 extends to its global action in many asymmetric cell divisions. In Aim 2, we will develop and implement high-resolution, high-throughput approaches to identifying causal genes underlying variation in the requirement for the major endoderm regulatory inputs. We will test candidate genes for modulation of the SKN-1-dependent activation of the endoderm GRN. In Aim 3, we will analyze the molecular basis for transgenerational inheritance (TEI) of GRN output. We will assess the stages in the endoderm GRN that are modulated by this TEI and test the hypothesis that epigenetic differences in SKN-1 requirement between selected isotypes extends to other regulatory inputs. We will test the hypothesis that TEI results from differences in chromatin states of endoderm genes and that differential expression of non-coding RNAs and endoderm regulatory genes is associated with TEI. Findings from this research will help to illuminate mechanisms of birth defects and can provide a paradigm for understanding relationships between an individual’s genotype and responsiveness to pharmacological agents, of importance to advancing precision medicine. They will also reveal factors that alter the outcome of Wnt signaling, a major regulatory mechanism associated with many cancers.