Cell-type Specific Strategies for Genome Maintenance

染色体断裂对细胞来说格外危险，因为一次断裂就可能导致数百个基因同时丢失。因此，所有生物体都可以检测到甚至是单个双链断裂(DSB)，启动一种反应，包括诱导修复、阻止细胞分裂、改变发育命运，在某些情况下还包括程序性细胞死亡(PCD)。这些反应因生物和细胞类型的不同而不同。损伤反应的细胞类型特异性在任何系统中都没有得到很好的表征，尽管它很重要。最近的进展使同时测量每个基因转录水平的变化成为可能。植物对DSB表现出强大的转录反应，涉及到数千个基因的诱导或抑制。新技术还可以从特定类型的细胞中提纯细胞核。结合“转录”和表型分析将增加对转录调控在双链球菌修复和耐受中的作用的理解。在植物内经历PCD反应的一小部分专门细胞中的转录反应将与周围细胞的转录反应进行比较，尽管经历了同样的DNA破坏处理，但周围的细胞没有死亡。这一方法将有助于识别Break诱导的细胞程序性死亡所需的基因，为植物PCD诱导的早期和后期阶段提供对这一过程的机制和标记的见解。一些转录因子也是由染色体断裂诱导的。利用这些调控基因中有缺陷的突变体，将研究反应中的亚程序及其生物学意义。最后，使这种反应在植物中选择性有利的染色体断裂的自然来源尚不清楚。最近的工作表明，种子在储存过程中经历的干燥和在酸性、富含金属的土壤中生长所引起的损害都会引起上述转录反应，这一假说将得到进一步的研究。广泛的影响：该项目将有助于理解植物细胞周期调节和细胞程序性死亡的机制。对植物基因组稳定性的新分析和对植物中DSB潜在自然来源的探索应该会产生与农业相关的发现。本科生、研究生和博士后学者将通过这笔赠款获得就业和培训，并将从非常多样化的人群中招聘。

Broken chromosomes are exceptionally dangerous to the cell, as a single break can result in the simultaneous loss of hundreds of genes. For this reason, all organisms can detect even a single double-strand break (DSB), initiating a response that includes the induction of repair, the arrest of cell division, changes in developmental fate, and in some cases programmed cell death (PCD). These responses vary from organism to organism, and with cell type. The cell-type specificity of damage response has not been well characterized in any system, in spite of its importance. Recent advances make it feasible to simultaneously measure changes in the transcription levels of every gene. Plants exhibit a robust transcriptional response to DSBs, involving the induction or suppression of thousands of genes. New technologies also allow purification of nuclei from specific cell types. Combining "transcriptomic" and phenotypic analysis will increase understanding of the role of transcriptional regulation in the repair and tolerance of DSBs. The transcriptional response in a small and specialized population of cells within the plant which undergo PCD in response to breaks will be compared to that of surrounding cells that do not die in spite of experiencing the same DNA-damaging treatment. This approach will allow identification of genes required for break-induced programmed cell death, providing insights into the mechanism of the process and markers for early and late steps in the induction of PCD in plants. Several transcription factors are also induced by chromosomal breaks. Using mutants defective in these regulatory genes, the subroutines within the response and their biological significance will be investigated.Finally, the natural source of chromosomal breaks that makes this response selectively advantageous in plants is unknown. Recent work indicates that both the desiccation experienced during seed storage and damage induced by growth in acidic, metal-rich soils provoke the transcriptional response described above, and this hypothesis will be investigated further.Broader impacts: This project will contribute to the understanding of cell cycle regulation and the mechanisms of programmed cell death in plants. Novel assays for genomic stability in plants and exploration of potential natural sources of DSBs in plants should result in findings of relevance to agriculture. Undergraduates, a graduate student, and a postdoctoral scholar will be employed and trained through this grant, and will be recruited from a very diverse population.