Identifying a transcriptional program that regulates compensatory proliferation

识别调节代偿性增殖的转录程序

• 批准号: 8737003
• 负责人: Joy Hart Meserve
• 金额: $ 3.24万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737003
• 关键词: Adult; Age; Aging; Animals; Apoptosis; Apoptotic; Bioinformatics; Biological Assay; Candidate Disease Gene; Cell Count; Cell Cycle; Cell Cycle Regulation; Cell division; Cell physiology; Cells; Development; Disease; Disease Progression; Drosophila eye; Drosophila genus; Drosophila melanogaster; Eye; Fluorescence-Activated Cell Sorting; Gene Expression Profile; Gene Targeting; Genes; Genetic; Homeostasis; Homologous Gene; Human; Inflammation; Knowledge; Lead; Life; Mitotic; Molecular; Molecular Analysis; Natural regeneration; Organ failure; Organism; Pathway interactions; Phenotype; Play; Population; Process; Proliferating; Proteins; RNA Interference; Regulatory Pathway; Research; Role; Signal Pathway; Signal Transduction; Stem Cell Development; Testing; Time; Tissues; Transcriptional Regulation; Undifferentiated; Vertebrates; cell growth regulation; cell injury; fly; genetic analysis; insight; knock-down; precursor cell; prevent; programs; public health relevance; regenerative; repaired; research study; response; stem cell population; tissue regeneration; tool; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): All animals undergo damage as they develop and age, and many organisms have evolved mechanisms to respond to and repair this damage. One such mechanism is compensatory proliferation, in which cells that are dying due to damage will mitogenically signal surrounding cells to increase proliferation and replace lost cells. This process is heavily utilized in damaged precursor cell populations during development and stem cells during adult life. However, many post-mitotic cells appear unable to re-enter the cell cycle and proliferate in response to damage, which hinders regeneration of adult tissues; the reasons for this inability are not known. In the eye precursor tissue of the fruit fly Drosophila melanogaster, there is a population of post-mitotic, undifferentiated cells that are able to undergo compensatory proliferation. How these cells are able to overcome negative cell cycle regulation and re-enter the cell cycle is currently unknown. Elucidating the genes involved in this process is important for understanding how to stop and perhaps reverse many of the phenotypes associated with aging. For example, tissues often accumulate damage with age as their regenerative abilities decrease. If the genes required for compensatory proliferation can be stimulated in these tissues, regeneration may be induced. Furthermore, damage often leads to inflammation and hyperproliferation, which contribute to disease development. Therefore, blocking genes involved in compensatory proliferation may prevent the accumulation of excess tissue and disease progression. This proposal aims to identify the genes involved in compensatory proliferation in the Drosophila post-mitotic eye precursor population, with the hypothesis that a unique transcriptional program is necessary for this process since a requirement for transcriptional regulation has already been shown. I will test this hypothesis by characterizing the transcriptional profile in these cells using fluorescence-activated cell sorting (FACS) to isolate the compensatory proliferating population and RNA-seq to identify the transcriptome. Genes that are highly expressed in these cells and not in their non-proliferating counterparts are likely to be involved in compensatory proliferation. In addition to providing candidate genes that may be required during compensatory proliferation, the results from these RNA-seq experiments will provide a hypothetical molecular network for compensatory proliferation. The second complementary approach will utilize RNAi to identify transcription factors required for compensatory proliferation. We will identify these necessary genes by individually knocking down expression of all Drosophila transcription factors, the majority of which have vertebrate homologs; genes that are required for compensatory proliferation in developing eyes subjected to damage will produce rough, small adult eyes due to a decrease in the number of cells present. Any additional candidates from the RNA-seq experiments will also be tested using this assay. Further genetic and molecular analysis will be performed to characterize the interactions between multiple pathways. The results from these experiments will provide a comprehensive view of compensatory proliferation and will provide candidate genes that may be involved in regeneration and disease in vertebrates.

描述（由申请人提供）：所有动物在发育和衰老过程中都会遭受损伤，许多生物体已经进化出应对和修复这种损伤的机制。其中一种机制是补偿性增殖，其中因损伤而死亡的细胞将向周围细胞发出有丝分裂信号，以增加增殖并替换丢失的细胞。这一过程在发育过程中受损的前体细胞群和成年过程中的干细胞中被大量利用。然而，许多有丝分裂后细胞似乎无法重新进入细胞周期并因损伤而增殖，这阻碍了成体组织的再生；造成这种情况的原因尚不清楚。在果蝇果蝇的眼睛前体组织中，有一群能够进行代偿性增殖的有丝分裂后未分化细胞。目前尚不清楚这些细胞如何克服负细胞周期调节并重新进入细胞周期。阐明与此相关的基因 这个过程对于理解如何阻止甚至逆转许多与衰老相关的表型非常重要。例如，组织通常会随着年龄的增长而累积损伤，因为它们的再生能力会下降。如果可以在这些组织中刺激代偿性增殖所需的基因，则可以诱导再生。此外，损伤常常导致炎症和过度增殖，从而导致疾病的发展。因此，阻断参与代偿性增殖的基因可能会阻止多余组织的积累和疾病进展。该提案旨在鉴定果蝇有丝分裂后眼前体群体中参与代偿性增殖的基因，并假设这一过程需要一个独特的转录程序，因为转录调控的要求已经被证明。我将通过使用荧光激活细胞分选来表征这些细胞中的转录谱来检验这个假设 (FACS) 分离补偿性增殖群体，并通过 RNA-seq 鉴定转录组。在这些细胞中而不是在其非增殖对应细胞中高度表达的基因可能参与代偿性增殖。除了提供补偿性增殖期间可能需要的候选基因之外，这些RNA-seq实验的结果还将提供补偿性增殖的假设分子网络。第二种补充方法将利用 RNAi 来识别代偿性增殖所需的转录因子。我们将通过单独敲除所有果蝇转录因子的表达来识别这些必要的基因，其中大多数转录因子具有脊椎动物同源物；由于细胞数量减少，发育中的眼睛受到损伤时补偿性增殖所需的基因会产生粗糙、小的成年眼睛。 RNA-seq 实验中的任何其他候选者也将使用此测定法进行测试。将进行进一步的遗传和分子分析，以表征多种途径之间的相互作用。这些实验的结果将提供补偿性增殖的全面视图，并将提供可能参与脊椎动物再生和疾病的候选基因。