The pathophysiological relevance of the histone variant H2A.J in radiation-induced, premature senescence

放射治疗后的长期副作用可以追溯到功能失调的衰老细胞的积累和组织特异性干细胞增殖能力的降低；然而，迄今为止，其发病机制尚不清楚。在我们的初步研究中，我们鉴定了新的组蛋白变异体H2A.J，它与染色质的整合随着电离辐射的增加而增加，并与细胞衰老有关。本研究项目的目的是研究组蛋白变异体H2A.J在体外和体内与辐射诱导的早衰有关的重要性。通过光镜和电子显微镜对H_2A.J+/+和H_2A.J-/-成纤维细胞进行比较研究，研究辐射诱导的DNA损伤、局部染色质修饰和随后染色质结构的整体重组之间的分子生物学关系。此外，应使用多参数流式细胞术对染色质组织和核完整性的改变进行结构和功能表征。应用高分辨率、精确的全细胞核三维重建，揭示照射后H_2A.J的染色质整合与染色质功能重组之间的联系。此外，H_2A_J的功能将在照射过程中和照射后皮肤的复杂组织结构中进行研究。毛囊及其隆起区位于小鼠的表皮，为分析表皮干细胞的分化过程提供了一个理想的模型。为了考察对组织动态平衡的影响，不同干细胞群体中辐射诱导的DNA损伤应与H_2A.J染色质整合相关，并分别与增殖、分化、凋亡和衰老相对照。应在转基因H_2A.J-/-小鼠身上检查H_2A.J在表皮中的特定功能。具体地说，丢失的H2A.J表达如何影响辐射后的过早衰老或表皮干细胞的不及时分化。BrdU标记的使用将捕捉干细胞群体的分裂速率和自我更新，并结合H2A.J检测，将与过早分化进行核对。由于干细胞从其利基迁移的增加导致毛囊的长期再生能力降低，因此需要分析毛发生长和着色的功能终点。总体而言，组蛋白变异体H_2A.J分别在辐射诱导、过早衰老和表皮干细胞的过早分化中的病理生理学相关性有待研究，以揭示开发纠正药物治疗的可能起点。

The long term side effects following radiotherapy can be traced back to the accumulation of dysfunctional senescent cells combined with the reduced proliferative potential of tissue-specific stem cells; to date the pathomechanisms however remain unclear. In our preliminary studies, we have characterized the novel histone variant H2A.J, whose integration into chromatin increases following ionizing radiation and additionally is associated with cellular senescence.The aim of this research project is to investigate the importance of the histone variant H2A.J in relation to radiation-induced, premature senescence both in-vitro and in-vivo. The molecular biological relationship between radiation-induced DNA damage, local chromatin modifications and subsequent global reorganization of the chromatin architecture is to be investigated using comparative studies between H2A.J+/+ and H2A.J-/- fibroblasts through the means of light- and electron microscopy. Additionally, the alterations in chromatin organization and nuclear integrity shall be structurally and functionally characterized using multi-parametric flow cytometry. The application of high-resolution, precise 3D reconstruction of the entire cell nucleus shall be used to uncover the connections between the chromatin integration of H2A.J following irradiation and the functional reorganization of chromatin.Furthermore, the functionality of H2A.J is to be investigated in the complex tissue configuration of the skin, during and after irradiation. Located in the murine epidermis is the hair follicle and its bulge region which presents an ideal model for the analysis of the differentiation processes of epidermal stem cells. To examine the effect on tissue homeostasis, the radiation-induced DNA damage in different stem cell populations shall be correlated with the chromatin integration of H2A.J and collated with proliferation, differentiation, apoptosis and senescence respectively. Analysis of the specific function of H2A.J in the epidermis shall be inspected in transgenic H2A.J-/- mice. In specific, how the missing H2A.J expression influences premature senescence or, as the case may be, the untimely differentiation of epidermal stem cells, following irradiation. The use of BrdU labeling will capture the division rate, as well as, the self-renewal of the stem cell population and, combined with H2A.J detection, will be collated with premature differentiation. As increased migration of stem cells from their niche leads to a reduced regeneration capacity of the hair follicle in the long term, the functional endpoint of hair growth and coloring shall be analyzed.Overall, the pathophysiological relevance of the histone variant H2A.J in radiation-induced, premature senescence and the untimely differentiation of epidermal stem cells respectively, is to be investigated to uncover possible starting points for the development of a corrective pharmacological therapy.