CD25 (IL2RA) 是激活的人类自然杀伤细胞中 PRDM1 的直接转录靶点,Blood

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CD25 (IL2RA) 是激活的人类自然杀伤细胞中 PRDM1 的直接转录靶点
Blood ( IF 21.0 ) Pub Date : 2018-11-29 , DOI: 10.1182/blood-2018-99-113460
Burcu Akman _{1,

2} , Deniz Kursun _{1,

2} , Tevfik Hatipoglu _{1,

2} , Xiaozhou Hu _{1,

2} , Wing C Chan ₃ , Can Kucuk _{2,

4}

Affiliation

摘要背景：PRDM1 是一种调节 B 和 T 淋巴细胞分化和/或稳态的转录因子。它还可能在自然杀伤 (NK) 细胞稳态中发挥作用，并且在 NK 细胞衍生的淋巴瘤中经常失活。PRDM1的许多靶基因已被报道；然而，对 NK 细胞中 PRDM1 直接靶向的基因的了解甚少。IL2 受体信号传导对 NK 细胞活化的许多特征（包括持续增殖和存活）至关重要。在这里，我们评估了参与 NK 细胞活化的关键基因 CD25 (IL2Rα) 在活化的人 NK 细胞中是否被 PRDM1 转录抑制。方法：将 ChIP-Seq 应用于使用涉及基因工程 NK 细胞靶细胞（即 K562-Cl9-mb21) 来确定 PRDM1 占据的基因组位置。CD25 中的 PRDM1 结合位点筛选是使用两个不同的 ChIP-Seq 峰值调用程序计算执行的。用共表达 PRDM1α 和 GFP 的逆转录病毒构建体转导两种恶性 NK 细胞系（NK92 和 KHYG1）。转导后 48 小时，用 DNA 微阵列、RNA-Seq 和 RT-qPCR 在 GFP 分选的 NK 细胞上评估 CD25 转录表达。CD25 表达在 NK92 和 KHYG1 细胞系中被两种不同的 shRNA 稳定地敲低。CD25 在通过与 K562-Cl9-mb21 共培养激活的原代人 NK 细胞中异位表达。分别对 CD25 shRNA 转导的 NK 细胞系或在限制 IL2 浓度下具有异位 CD25 表达的原代 NK 细胞进行 GFP 竞争测定。结果：用 CisGenome 和 MACS 软件在活化的原代 NK 细胞中检测到 PRDM1 占据 CD25 的第一个内含子。通过 ChIP-qPCR 在正常活化的 NK 细胞中进一步验证 PRDM1 与 CD25 的直接结合。两个 PRDM1α 转导的 PRDM1 非表达 NK 细胞系通过 DNA 微阵列和 RNA-Seq 显示出对 CD25 的显着转录抑制，其抑制与 RT-qPCR 交叉验证。CD25 的稳定敲低抑制了在限制 IL2 浓度下培养的两种 NK 细胞系的生长。相比之下，CD25 的异位表达导致体外培养的原代 NK 细胞的阳性选择。结论：总而言之，这些结果确立了 CD25 作为人 NK 细胞中 PRDM1 的直接转录靶标。这些观察结果为 PRDM1 在终止 NK 细胞活化和生长中的作用提供了额外的支持，当它被解除管制时对肿瘤转化或 NK 细胞功能有影响。披露没有需要声明的相关利益冲突。

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CD25 (IL2RA) Is a Direct Transcriptional Target of PRDM1 in Activated Human Natural Killer Cells

Abstract Background: PRDM1 is a transcription factor that regulates differentiation and/or homeostasis of B and T lymphocytes. It may also play a role in natural killer (NK) cell homeostasis, and it is frequently inactivated in NK cell-derived lymphomas. Many target genes of PRDM1 have been reported; however, there is little insight into genes directly targeted by PRDM1 in NK cells. IL2 receptor signaling is crucial for many features of NK cell activation including sustained proliferation and survival. Here we evaluated whether CD25 (IL2Rα), a critical gene involved in NK cell activation, is transcriptionally repressed by PRDM1 in activated human NK cells. Methods: ChIP-Seq was applied on normal primary NK cells activated using a special co-culture system that involved genetically engineered NK cell target cells (i.e. K562-Cl9-mb21) to determine genomic locations occupied with PRDM1. PRDM1 binding site screen in CD25 was computationally performed using two different ChIP-Seq peak callers. Two malignant NK cell lines (NK92 and KHYG1) were transduced with a retroviral construct that co-expresses PRDM1α and GFP. CD25 transcription expression was evaluated with DNA microarray, RNA-Seq and RT-qPCR on GFP-sorted NK cells 48h post-transduction. CD25 expression was stably knocked-down with two different shRNAs in NK92 and KHYG1 cell lines. CD25 was ectopically expressed in primary human NK cells activated through coculturing with K562-Cl9-mb21. GFP competition assays were performed on CD25 shRNA-transduced NK cell lines or primary NK cells with ectopic CD25 expression in limiting IL2 concentrations, respectively. Results: PRDM1 occupancy on the 1st intron of CD25 was detected with CisGenome and MACS softwares in activated primary NK cells. Direct binding of PRDM1 on CD25 was further validated by ChIP-qPCR in normal activated NK cells. Two PRDM1α-transduced PRDM1-nonexpressing NK cell lines showed significant transcriptional repression of CD25 by DNA microarray and RNA-Seq, the repression of which was cross-validated with RT-qPCR. Stable knock-down of CD25 inhibited growth of two NK cell lines cultured in limiting IL2 concentrations. By contrast, ectopic expression of CD25 resulted in positive selection of ex vivo cultured primary NK cells. Conclusions: Altogether these results establish CD25 as a direct transcriptional target of PRDM1 in human NK cells. These observations provide additional support for the role of PRDM1 in termination of NK cell activation and growth, with implications on neoplastic transformation or NK cell function when it is deregulated. Disclosures No relevant conflicts of interest to declare.

更新日期：2018-11-29

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