Role of Histon H2A deubiquitinase 2A-DUB/Mysm1 in lymphoid development and interaction with p53-mediated apoptotic programs

多能祖细胞的特化细胞群的顺序分化需要组蛋白修饰和染色质重塑酶与序列特异性DNA结合转录因子（TF）在更大的多蛋白复合物中协同作用，以协调的方式激活和沉默谱系特异性基因。组蛋白H2A去泛素酶2A-DUB/Mysm1是一种表观遗传调节因子，在淋巴细胞和皮肤中高度表达，可能在造血和皮肤发育的不同阶段实现几种关键功能：在早期淋巴细胞发育过程中，T细胞受体（TCR）和免疫球蛋白（Ig）基因的有效体细胞重组所需的dna损伤反应（DDR）。2. 在淋巴细胞和皮肤细胞分化过程中调控序列特异性tf对DNA的通路。3. 活性氧（ROS）和辐照诱导的DDR。因此，2A-DUB/Mysm1的H2A去泛素化可能参与调节对自身免疫、淋巴瘤和癌症的保护作用。由于2A-DUB/Mysm1基因工程化缺陷的小鼠（Mysm1 KO小鼠）在T细胞和皮肤发育方面存在许多缺陷——包括严重的胸腺发育不全以及外周T细胞活化改变、皮肤萎缩和早衰——我们建议使用遗传学、免疫学和分子生物学方法来表征Mysm1在T细胞、皮肤和相关疾病中的作用。基于申请人初步数据确定Ink4/ARF通路是Mysm1缺陷胸腺细胞和表皮细胞凋亡增加和增殖率降低的潜在介质，我们将在新生成的p53-/-Mysm1-/-和Cdkn2a/ARF-/- Mysm1-/-双ko （DKO）小鼠中研究并验证2A-DUB/Mysm1与p19ARF/Ink4a启动子的相互作用。我们将在Lck-Cre x Mysm1flox/flox小鼠以及p53-/-Mysm1-/- DKO和H-Y TCR转基因小鼠中探讨Mysm1组蛋白H2A去泛素化在胸腺细胞发育和选择中的作用。Mysm1在T细胞和皮肤细胞群体中的其他分子相互作用和结合伙伴将通过基于芯片的方法进行验证。最终，mysm1缺陷对T细胞库或T细胞和皮肤细胞遗传不稳定性的潜在改变的影响将在自身免疫和肿瘤模型中通过翻译方法进行探讨。本研究旨在更好地了解表观遗传机制如何与谱系特异性tf和分化程序相互作用以调节T细胞和皮肤发育，以及表观遗传如何有助于预防自身免疫和癌症。

Sequential differentiation of specialized cell populations from pluripotent progenitors requires the collaboration of histone modifying and chromatin remodeling enzymes with sequence-specific DNA binding transcription factors (TF) in larger multi-protein complexes to activate and silence lineage-specific genes in a coordinated manner. Histone H2A deubiquitinase 2A-DUB/Mysm1, an epigenetic regulator highly expressed in lymphocytes and the skin, potentially fulfills several critical functions at different stages of hematopoietic and skin development: 1. In the DNA-damage response (DDR) required for efficient somatic recombination of T cell receptor (TCR) and immunoglobin (Ig) genes during early lymphocyte development. 2. In regulating access of sequence-specific TFs to DNA during differentiation of lymphocytes and skin cells. 3. In the DDR induced by reactive oxygen species (ROS) and irradiation. Therefore, H2A deubiquitination by 2A-DUB/Mysm1 might be involved in regulating protection from autoimmunity, lymphoma and cancer. Because mice with an engineered deficiency in 2A-DUB/Mysm1 (Mysm1 KO mice) have a number of defects in T cell and skin development - including severe thymus hypoplasia along with altered activation of peripheral T cells, skin atrophy and premature aging -, we propose here to characterize the role of Mysm1 in T cells, the skin, and in associated diseases using genetic, immunological and molecularbiological approaches. Based on the applicants preliminary data identifying the Ink4/ARF pathway as potential mediator of increased apoptosis and reduced proliferation rates in Mysm1-deficient thymocytes and epidermal cells, the interplay of 2A-DUB/Mysm1 with the p19ARF/Ink4a promoter will be investigated and verified in our newly generated p53-/-Mysm1-/- and Cdkn2a/ARF-/- Mysm1-/- double-KO (DKO) mice. The role of histone H2A deubiquitination by Mysm1 in thymocyte development and selection will be explored in Lck-Cre x Mysm1flox/flox mice as well as in p53-/-Mysm1-/- DKO and H-Y TCR transgenic mice. Additional molecular interactions and binding partners of Mysm1 in T cell and skin cell populations will be verified by ChIP-based approaches. Ultimately, the impact of potential alterations in the T cell repertoire or in genetic instability in T cells and skin cells as a consequence of Mysm1-deficiency will be explored in autoimmune and tumor models in a translational approach. This investigation aims at providing a better understanding of how epigenetic mechanisms interact with lineage-specific TFs and differentiation programs to regulate T cell and skin development and how epigenetics contribute to the prevention of autoimmunity and cancer.