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

从多能祖细胞中顺序分化特化细胞群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作，以协调的方式激活和沉默谱系特异性基因。组蛋白H2 A去泛素化酶2A-DUB/Mysm 1是一种在淋巴细胞和皮肤中高度表达的表观遗传调节因子，在造血和皮肤发育的不同阶段可能实现几个关键功能：1.在早期淋巴细胞发育期间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）中。2.在淋巴细胞和皮肤细胞分化过程中调节序列特异性TF对DNA的访问。3.在由活性氧（ROS）和辐射诱导的DDR中。因此，2A-DUB/Mysm 1的H2 A去泛素化可能参与调节对自身免疫、淋巴瘤和癌症的保护。由于2A-DUB/Mysm 1基因工程缺陷小鼠（Mysm 1基因敲除小鼠）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沿着外周T细胞活化改变、皮肤萎缩和过早衰老-因此，我们在此建议使用遗传学、免疫学和分子生物学方法来表征Mysm 1在T细胞、皮肤和相关疾病中的作用。根据申请人初步数据，确定Ink 4/ARF途径是Mysm 1缺陷型胸腺细胞和表皮细胞中细胞凋亡增加和增殖速率降低的潜在介体，将在我们新产生的p53-/-Mysm 1-/-和Cdkn 2a/ARF-/-Mysm 1-/-双KO（DKO）小鼠中研究和验证2A-DUB/Mysm 1与p19 ARF/Ink 4a启动子的相互作用。将在Lck-Cre x Mysm 1flox/flox小鼠以及p53-/-Mysm 1-/- DKO和H-Y TCR转基因小鼠中探索Mysm 1对组蛋白H2 A去泛素化在胸腺细胞发育和选择中的作用。T细胞和皮肤细胞群中Mysm 1的其他分子相互作用和结合伴侣将通过基于ChIP的方法进行验证。最终，在T细胞库的潜在改变或在T细胞和皮肤细胞的遗传不稳定性作为Mysm 1缺陷的结果的影响将在自身免疫和肿瘤模型中的翻译方法进行探讨。这项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，以及表观遗传学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.