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The spatial regulation of genetic and epigenetic integrity in Embryonic Stem cells

胚胎干细胞遗传和表观遗传完整性的空间调控

基本信息

批准号：
BB/X016404/1
负责人：
Evi Soutoglou
金额：
$ 100.1万
依托单位：
University of Sussex
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2023
资助国家：
英国
起止时间：
2023 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FX016404%2F1
关键词：
spatial regulation genetic epigenetic integrity

项目摘要

DNA is unstable and it can bear different types of lesions. We study breaks which affect both strands of DNA, the Double Strand Breaks (DSBs). DSBs can be repaired in an error free manner by copying the information from the sister chromatin using Homologous recombination. But they can also lead to deletions which can be either small if Non Homologous End Joining (NHEJ) is used, or larger if the ends are re-joined using the microhomology found in the two sides of the break, via Microhomology Mediated End Joining (MMEJ). Deletions signatures from these pathways have been found in cancer genomes. DNA is in the form of chromatin and chromatin is not linear, but it is folded in three dimensions and associates with different nuclear compartments which further specify chromatin characteristics. A DSB also leads to extensive remodelling of the chromatin structure around the break with histone exchange and alterations of histone modifications. Currently, it is unknown to what extend the epigenome is restored after DNA damage. Permanent genetic and epigenetic scars can alter gene expression profiles and are hallmark of cancer. If these scars occur in embryos or in stem cells, they can alter cell identity and the reprogramming potential. Our work together with work from other labs demonstrated that active chromatin is more prone to error-free repair and that compacted chromatin is more to MMEJ/NHEJ. Chromatin and 3D genome organization is cell type specific and dramatically changes during differentiation. In addition, stem cells bear a very unique chromatin feature which in called bivalency in which active and inactive chromatin marks exist together at the same nucleosome and decorates developmentally regulated promoters and protects them from DNA methylation observed in cancers.It is currently unknown:1. Do DNA repair pathways adapt to changes in chromatin 3D genome organization to confer a cell type specificity in DNA repair fidelity? 2. Is the epigenome fully restored after DNA damage?In this proposal we will use mouse Embryonic Stem cells and study the spatial regulation of DNA repair fidelity and how this changes upon differentiation in different lineages . Then we will investigate the mechanisms controlling DNA repair fidelity at each chromatin state. Finally, we will ask whether the changes at the chromatin structure are fully restored after DNA damage and study the impact of genetic and epigenetic scars in stem cell identity.Our proposal will elucidate the complex relationship between genome and epigenome integrity and its link to mutagenesis and cell identify. Recently, there has been considerable effort in developing genome editing methods which are based on generation of DNA lesions by CRISP Cas nucleases used in this proposal. Therefore, our results will be very valuable for medical and research purposes as detailed understanding of genome editing effectiveness and particularly fidelity and precision in embryonic stages and how this altered in adult tissues, is of paramount importance for correcting disease mutations at stem cells which can then differentiated in the lab to the tissue which is affected by a disease.

DNA是不稳定的，它可以承受不同类型的病变。我们研究影响DNA双链的断裂，即双链断裂（DSB）。DSB可以通过使用同源重组从姐妹染色质复制信息而以无错误的方式修复。但是它们也可以导致缺失，如果使用非同源末端连接（NHEJ），则缺失可以是小的，或者如果通过微同源介导的末端连接（MMEJ）使用在断裂两侧发现的微同源重新连接末端，则缺失可以更大。在癌症基因组中已经发现了来自这些途径的缺失签名。DNA是染色质的形式，染色质不是线性的，但它是三维折叠的，并与不同的核隔室相关联，进一步指定染色质特征。DSB还导致断裂周围的染色质结构的广泛重塑，伴随组蛋白交换和组蛋白修饰的改变。目前，尚不清楚DNA损伤后表观基因组恢复到何种程度。永久性遗传和表观遗传疤痕可以改变基因表达谱，是癌症的标志。如果这些疤痕发生在胚胎或干细胞中，它们可以改变细胞的身份和重新编程的潜力。我们的工作与其他实验室的工作一起证明，活性染色质更容易进行无错误修复，致密染色质更容易进行MMEJ/NHEJ。染色质和3D基因组组织是细胞类型特异性的，并且在分化期间发生显著变化。此外，干细胞具有非常独特的染色质特征，称为二价，其中活性和非活性染色质标记一起存在于相同的核小体上，并装饰发育调节的启动子，并保护它们免受癌症中观察到的DNA甲基化。DNA修复途径是否适应染色质3D基因组组织的变化，以赋予DNA修复保真度的细胞类型特异性？2. DNA损伤后表观基因组是否完全恢复？在这项提案中，我们将使用小鼠胚胎干细胞，研究DNA修复保真度的空间调节以及在不同谱系中分化时如何变化。然后，我们将研究在每个染色质状态下控制DNA修复保真度的机制。最后，我们将探讨DNA损伤后染色质结构的变化是否完全恢复，并研究遗传和表观遗传疤痕对干细胞身份的影响。我们的建议将阐明基因组和表观基因组完整性之间的复杂关系及其与诱变和细胞鉴定的联系。最近，在开发基因组编辑方法方面已经做出了相当大的努力，该方法基于通过本提案中使用的CRISP Cas核酸酶产生DNA损伤。因此，我们的研究结果对于医学和研究目的非常有价值，因为详细了解基因组编辑的有效性，特别是胚胎阶段的保真度和精确度，以及这在成人组织中如何改变，对于纠正干细胞的疾病突变至关重要，然后干细胞可以在实验室中分化为受疾病影响的组织。