Programming of gene expression is essential for cellular differentiation and function. This is accomplished through a complex system of epigenetic regulatory mechanisms that include post‐translational modifications to the histones that pack and order DNA in the nucleus. Among these modifications, mono‐, di‐, and tri‐methylation of histone H3 at lysine 79 (H3K79me) are mediated by a histone lysine methyltransferase known as Disruptor of telomeric silencing 1‐like (DOT1L).¹ DOT1L has been implicated in diverse biological processes including embryonic development, DNA damage response and meiotic checkpoint control,² and is a promising drug target for the treatment of mixed lineage leukemia (MLL)‐rearranged leukemia where MLL fusion proteins recruit DOT1L to MLL target genes.² DOT1L has been implicated in both gene activation³ and repression⁴ in different contexts. Despite the emerging roles for DOT1L in various cell types and diseases, its function in the immune system was, until recently, largely uncharacterised. In two recent articles, studies by Sebastian Scheer and colleagues⁵ and Liam Kealy and colleagues⁶ define critical regulatory functions for DOT1L in T and B lymphocytes. Because DOT1L deficiency leads to embryonic lethality in mice,⁷ both studies utilise conditional knockout (KO) mouse models to investigate DOT1L function in specific populations of T and B lymphocytes.

Scheer et al. show that DOT1L is an important regulator of T‐cell development by crossing Cd4‐Cre transgenic mice with Dot1l^fl/fl mice to generate mice lacking DOT1L in T cells.⁵ These studies were prompted by a recent discovery by the same group that identified DOT1L as a regulator of T‐cell function.⁸ Genetic DOT1L deficiency in T cells resulted in a large reduction in H3K79me2 in both CD4⁺ and CD8⁺ T cells. CD4⁺ T‐cell numbers were markedly reduced in the thymus and spleen and exhibited both increased apoptosis and increased activation via the T‐cell receptor (TCR). DOT1L therefore appears to be a central regulator of both T‐cell function and homeostasis. The authors also found spontaneous accumulation of T cells expressing the activation marker CD44, but further analysis showed that this phenotype was due to lymphopenia, rather than an intrinsic property of T cells lacking DOT1L. Lymphopenia was also found by others to underlie phenotypical changes in T cells that can be induced by CD4‐Cre‐driven deletion of the important transcription factor KLF2,⁹ indicating that lymphopenia could be responsible for at least some of the phenotypical changes in T cells in mice lacking other important transcriptional regulators. Naïve CD4⁺ T cells can differentiate into several stable or semi‐stable subsets of T helper (Th) cells after antigen encounter, including interferon gamma (IFN‐γ)‐producing Th1 cells, interleukin (IL)‐4/5/13‐producing Th2 cells and IL‐17‐producing Th17 cells.¹⁰ Scheer et al. have found that DOT1L deficiency leads to elevated IFN‐γ expression in CD4⁺ T cells that is largely dependent on the Th1 master transcription factor T‐bet. Differentiated Th2 cells are normally resistant to upregulation of Th1 cell‐associated genes through epigenetic programming,^{11, 12} and the findings from Scheer et al. define DOT1L as another key player in the regulation of lineage stability. Specifically, DOT1L‐deficient Th2 and Th17 cells are able to be reprogrammed into cells expressing Th1‐associated genes including IFN‐γ. Because H3K79me2 has primarily been correlated with gene activation, the authors also considered the possibility that DOT1L might be required to promote expression of transcriptional regulators that inhibit the Th1 program. Indeed, highly expressed genes had more H3K79me2 and expression of several important immunoregulatory genes including Bach2 and Foxp3 were reduced in DOT1L‐deficient Th cells. This indicates that DOT1L could potentially control Th1 differentiation through repression of Th1 genes and by activation of negative regulators. Importantly, DOT1L deletion was found to impair Th2 responses in vivo, suggesting that DOT1L inhibition with small molecule inhibitors could be a viable therapeutic strategy to limit Th2 cell responses such as those observed in allergic diseases.

Appearing back‐to‐back in the issue of Cell Reports is an independent study from Kealy et al. that find a central role for DOT1L in the differentiation and function of B lymphocytes.⁶ The authors employ two different strategies for deletion of DOT1L and an extensive range of immunological challenges to convincingly define the involvement of DOT1L in the humoral immune response. Using Mb1‐Cre‐driven deletion of Dot1l^fl/fl, the authors found smaller spleens and a reduced population of B cells in both the spleen and bone marrow (BM) in mice lacking DOT1L in all B cells. To specifically delete DOT1L in mature B cells, the authors crossed Cd23‐Cre mice with Dot1l^fl/fl mice and show that DOT1L is critically required for the generation of antigen‐specific antibody responses after immunisation. Strikingly, DOT1L‐deficiency in mature B cells also resulted in the absence of memory B cells and decreased plasma cells in the BM following influenza virus infection, and DOT1L was found to be needed for an effective GC reaction and class‐switched antibody formation in both Th1‐ and Th2‐biased immune responses. Notably, DOT1L was found to be critical for both T‐cell‐dependent and T‐cell‐independent isotype‐switched antibody production. Neither proliferation nor apoptosis was altered in DOT1L‐deficient B cells, indicating that changes in these fundamental physiological processes were not causing the absence of isotype‐switched humoral immunity. Analysis of the transcriptome of DOT1L‐deficient B cells after immunisation identified reduced expression of several genes associated with B cell signaling and migration, prompting the authors to investigate the localisation of B cells in DOT1L‐deficient mice. BCL6 is a critical transcription factor that is important for GC formation,¹³ and during a normal immune response B cells expressing BCL6 are found within B cell follicles within days after immunisation. B cells lacking DOT1L were still found to express BCL6, but the BCL6‐expressing cells failed to localise inside the follicles. BCL6‐expressing B cells were instead primarily found around the outer edge of the follicle and in the marginal zone in the spleen. Together, these results define DOT1L as a central regulator of the humoral immune response.

The findings of the studies by Scheer et al.⁵ and Kealy et al.⁶ provide a thorough insight into the functions of DOT1L in B and T cells and greatly expand our understanding of how gene expression in lymphocytes is regulated. The studies indicate that DOT1L displays some degree of specificity in the gene programs that it controls, i.e. the Th1 program in CD4⁺ T cells and the differentiation of class‐switched antibody‐producing B cells, potentially by regulating B cell homing within lymphoid tissue. Within these differentiation programs, DOT1L could potentially repress and activate different genes simultaneously. Defining the exact way that DOT1L accomplishes this will require further investigation. It will also be important to determine whether histone methylation‐independent functions of DOT1L are responsible for mediating some of its functions.¹⁴ Finally, these studies raise important clinical questions including the extent to which inhibition of DOT1L with drugs designed to treat malignant tumors might also affect healthy B and T cells.

基因表达的编程对于细胞分化和功能是必不可少的。这是通过复杂的表观遗传调控机制系统实现的，其中包括对在细胞核中包装和排列 DNA 的组蛋白进行翻译后修饰。在这些修饰中，组蛋白 H3 在赖氨酸 79 (H3K79me) 处的单、二和三甲基化由组蛋白赖氨酸甲基转移酶介导，称为端粒沉默 1 样干扰因子 (DOT1L)。¹ DOT1L 涉及多种生物过程，包括胚胎发育、DNA 损伤反应和减数分裂检查点控制，²并且是治疗混合谱系白血病 (MLL) 重排白血病的有前途的药物靶标，其中 MLL 融合蛋白将 DOT1L 募集到 MLL 靶标基因。² DOT1L与不同背景下的基因激活³和抑制⁴都有关联。尽管 DOT1L 在各种细胞类型和疾病中发挥着新的作用，但直到最近，它在免疫系统中的功能还基本上没有特征。在最近的两篇文章中，Sebastian Scheer 及其同事⁵和 Liam Kealy 及其同事^{6 的研究}定义了 T 和 B 淋巴细胞中 DOT1L 的关键调节功能。由于 DOT1L 缺陷导致小鼠胚胎致死^7，两项研究均利用条件敲除 (KO) 小鼠模型来研究特定 T 和 B 淋巴细胞群体中的 DOT1L 功能。

谢尔等人。通过将Cd4- Cre 转基因小鼠与Dot1l ^fl/fl小鼠杂交以产生 T 细胞中缺乏 DOT1L 的小鼠，表明 DOT1L 是 T 细胞发育的重要调节剂。⁵这些研究受到同一小组最近的一项发现的启发，该发现将 DOT1L 确定为 T 细胞功能的调节剂。⁸ T 细胞中的遗传 DOT1L 缺陷导致 CD4 ⁺和 CD8 ⁺ T 细胞中 H3K79me2 的大量减少。CD4 ⁺胸腺和脾脏中的 T 细胞数量显着减少，并通过 T 细胞受体 (TCR) 显示出增加的细胞凋亡和激活增加。因此，DOT1L 似乎是 T 细胞功能和体内平衡的中央调节器。作者还发现表达激活标志物 CD44 的 T 细胞自发积累，但进一步分析表明，这种表型是由于淋巴细胞减少所致，而不是缺乏 DOT1L 的 T 细胞的内在特性。其他人还发现淋巴细胞减少是 T 细胞表型变化的基础，这种变化可以由 CD4-Cre 驱动的重要转录因子 KLF2 的缺失引起，⁹表明淋巴细胞减少可能是缺乏其他重要转录调节因子的小鼠中 T 细胞的至少一些表型变化的原因。初始 CD4 ⁺ T 细胞在遇到抗原后可分化为几个稳定或半稳定的 T 辅助 (Th) 细胞亚群，包括产生干扰素 γ (IFN-γ) 的 Th1 细胞、白细胞介素 (IL)-4/5/13-产生 Th2 细胞和产生 IL-17 的 Th17 细胞。¹⁰谢尔等人。已经发现 DOT1L 缺乏导致 CD4 ⁺ T 细胞中IFN-γ 表达升高，这在很大程度上取决于 Th1 主转录因子 T-bet。分化的 Th2 细胞通常通过表观遗传编程对 Th1 细胞相关基因的上调具有抗性，^{11, 12}以及 Scheer等人的发现。将 DOT1L 定义为血统稳定性调节中的另一个关键参与者。具体而言，DOT1L 缺陷型 Th2 和 Th17 细胞能够重编程为表达 Th1 相关基因（包括 IFN-γ）的细胞。由于 H3K79me2 主要与基因激活相关，作者还考虑了可能需要 DOT1L 来促进抑制 Th1 程序的转录调节因子的表达。事实上，高表达的基因有更多的 H3K79me2 和几个重要的免疫调节基因的表达，包括Bach2和Foxp3在 DOT1L 缺陷的 Th 细胞中减少。这表明 DOT1L 可能通过抑制 Th1 基因和激活负调节因子来控制 Th1 分化。重要的是，发现 DOT1L 缺失会损害体内Th2 反应，这表明用小分子抑制剂抑制 DOT1L 可能是一种可行的治疗策略，以限制 Th2 细胞反应，例如在过敏性疾病中观察到的反应。

在《细胞报告》一期中连续出现是 Kealy等人的一项独立研究。发现 DOT1L 在 B 淋巴细胞分化和功能中的核心作用。⁶作者采用了两种不同的 DOT1L 缺失策略和广泛的免疫学挑战，以令人信服地定义 DOT1L 参与体液免疫反应。使用Mb1 -Cre 驱动的Dot1l ^fl/fl缺失，作者发现在所有 B 细胞中缺乏 DOT1L 的小鼠的脾脏和骨髓 (BM) 中脾脏更小，B 细胞数量减少。为了特异性删除成熟 B 细胞中的 DOT1L，作者将Cd23- Cre 小鼠与Dot1l ^fl/fl小鼠并表明 DOT1L 是免疫后产生抗原特异性抗体反应所必需的。引人注目的是，成熟 B 细胞中的 DOT1L 缺陷也导致流感病毒感染后记忆 B 细胞的缺失和 BM 中的浆细胞减少，并且发现 DOT1L 是有效的 GC 反应和类别转换抗体形成所必需的Th1 和 Th2 偏向免疫反应。值得注意的是，DOT1L 被发现对 T 细胞依赖性和 T 细胞非依赖性同种型转换抗体的产生至关重要。DOT1L 缺陷型 B 细胞的增殖和凋亡均未发生改变，表明这些基本生理过程的变化并未导致同种型转换体液免疫的缺失。对免疫后 DOT1L 缺陷型 B 细胞转录组的分析发现与 B 细胞信号传导和迁移相关的几个基因的表达降低，促使作者研究 B 细胞在 DOT1L 缺陷型小鼠中的定位。BCL6 是一种关键的转录因子，对 GC 形成很重要，¹³和在正常免疫反应期间，在免疫后几天内在 B 细胞滤泡内发现表达 BCL6 的 B 细胞。仍发现缺乏 DOT1L 的 B 细胞表达 BCL6，但表达 BCL6 的细胞未能定位于毛囊内。相反，表达 BCL6 的 B 细胞主要出现在卵泡的外缘和脾脏的边缘区。总之，这些结果将 DOT1L 定义为体液免疫反应的中央调节器。

Scheer等人的研究结果。⁵和 Kealy等人。^{图 6}提供了对 B 和 T 细胞中 DOT1L 功能的全面了解，并极大地扩展了我们对淋巴细胞中基因表达如何调节的理解。研究表明 DOT1L 在它控制的基因程序中显示出一定程度的特异性，即 CD4 ^{+ 中}的 Th1 程序T 细胞和产生类别转换抗体的 B 细胞的分化，可能是通过调节淋巴组织内的 B 细胞归巢。在这些分化程序中，DOT1L 可能同时抑制和激活不同的基因。定义 DOT1L 实现此目的的确切方式将需要进一步调查。确定 DOT1L 的组蛋白甲基化独立功能是否负责介导其某些功能也很重要。¹⁴最后，这些研究提出了重要的临床问题，包括设计用于治疗恶性肿瘤的药物抑制 DOT1L 的程度也可能影响健康的 B 和 T 细胞。