The crucial role of the major histocompatibility complex (MHC) for the immune response to infectious diseases is well-known, but no information is available on the 3D nuclear organization of this gene-dense region in immune cells, whereas nuclear architecture is known to play an essential role on genome function regulation. We analyzed the spatial arrangement of the three MHC regions (class I, III and II) in macrophages using 3D-FISH. Since this complex presents major differences in humans and pigs with, notably, the presence of the centromere between class III and class II regions in pigs, the analysis was implemented in both species to determine the impact of this organization on the 3D conformation of the MHC. The expression level of the three genes selected to represent each MHC region was assessed by quantitative real-time PCR. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen modifies their expression level and their 3D organization. While the three MHC regions occupy an intermediate radial position in porcine macrophages, the class I region was clearly more peripheral in humans. The BAC center-to-center distances allowed us to propose a 3D nuclear organization of the MHC in each species. LPS/IFNγ activation induces a significant decompaction of the chromatin between class I and class III regions in pigs and between class I and class II regions in humans. We detected a strong overexpression of TNFα (class III region) in both species. Moreover, a single nucleus analysis revealed that the two alleles can have either the same or a different compaction pattern. In addition, macrophage activation leads to an increase in alleles that present a decompacted pattern in humans and pigs. The data presented demonstrate that: (i) the MHC harbors a different 3D organization in humans and pigs; (ii) LPS/IFNγ activation induces chromatin decompaction, but it is not the same area affected in the two species. These findings were supported by the application of an original computation method based on the geometrical distribution of the three target genes. Finally, the position of the centromere inside the swine MHC could influence chromatin reorganization during the activation process.

中文翻译：

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猪和人巨噬细胞中主要组织相容性复合物的 3D 核构象在细胞激活时发生变化

主要组织相容性复合物 (MHC) 对传染病免疫反应的关键作用是众所周知的，但没有关于免疫细胞中该基因密集区域的 3D 核组织的信息，而已知核结构在其中发挥着重要作用在基因组功能调控中发挥重要作用。我们使用 3D-FISH 分析了巨噬细胞中三个 MHC 区域（I、III 和 II 类）的空间排列。由于该复合体在人类和猪中存在重大差异，特别是在猪的 III 类和 II 类区域之间存在着丝粒，因此在这两个物种中进行了分析，以确定该组织对 MHC 3D 构象的影响。通过定量实时 PCR 评估代表每个 MHC 区域的三个基因的表达水平。研究了静息状态和脂多糖 (LPS) 激活状态，以确定对病原体的反应是否会改变其表达水平及其 3D 组织。虽然三个 MHC 区域在猪巨噬细胞中占据中间径向位置，但 I 类区域在人类中显然更外围。BAC 中心距使我们能够提出每个物种 MHC 的 3D 核组织。LPS/IFNγ 激活可诱导猪 I 类和 III 类区域之间以及人类 I 类和 II 类区域之间染色质的显着解压缩。我们在这两个物种中检测到 TNFα（III 类区域）的强烈过度表达。此外，单核分析表明，两个等位基因可以具有相同或不同的压缩模式。此外，巨噬细胞激活导致等位基因增加，在人类和猪中呈现出压缩模式。所提供的数据表明：（i）人类和猪的 MHC 具有不同的 3D 组织；(ii) LPS/IFNγ 激活会诱导染色质解压缩，但两个物种中受影响的区域不同。这些发现得到了基于三个目标基因的几何分布的原始计算方法的应用的支持。最后，猪 MHC 内着丝粒的位置可能会影响激活过程中的染色质重组。