OBJECTIVE Genetic variants in the region of tumor necrosis factor-induced protein 3-interacting protein 1 (TNIP1) are associated with autoimmune disease and reduced TNIP1 gene expression. The aim of this study was to define the functional genetic mechanisms driving TNIP1 hypomorphic expression imparted by the systemic lupus erythematosus-associated TNIP1 H1 risk haplotype. METHODS Dual luciferase expression and electrophoretic mobility shift assays were used to evaluate the allelic effects of 11 risk variants on enhancer function and nuclear protein binding in immune cell line models (Epstein-Barr virus [EBV]-transformed human B cells, Jurkat cells, and THP-1 cells), left in a resting state or stimulated with phorbol 12-myristate 13-acetate/ionomycin. HiChIP was used to define the regulatory 3-dimensional (3-D) chromatin network of the TNIP1 haplotype by detecting in situ long-range DNA contacts associated with H3K27ac-marked chromatin in EBV B cells. Then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression of genes within the 3-D chromatin network. RESULTS Bioinformatics analyses of 50 single-nucleotide polymorphisms on the TNIP1 H1 risk haplotype identified 11 non-protein-coding variants with a high likelihood of influencing TNIP1 gene expression. Eight variants in EBV B cells, 5 in THP-1 cells, and 2 in Jurkat cells exhibited various allelic effects on enhancer activation, resulting in a cumulative suppressive effect on TNIP1 expression (net effect of risk variants -7.14 fold, -6.80 fold, and -2.44 fold, respectively; n > 3). Specifically, in EBV B cells, only 2 variants (rs10057690 and rs13180950) exhibited allele-specific loss of both enhancer activity and nuclear protein binding (each P < 0.01 relative to nonrisk alleles). In contrast, the rs10036748 risk allele reduced binding affinities of the transcriptional repressors basic helix-loop-helix family member 40/differentially expressed in chondrocytes 1 (bHLHe40/DEC1) (P < 0.05 relative to nonrisk alleles) and CREB-1 (P not significant) in EBV B cells, resulting in a gain of enhancer activity (P < 0.05). HiChIP and qRT-PCR analyses revealed that overall transcriptional repression of the TNIP1 haplotype extended to the neighboring genes DCTN4 and GMA2, both of which also showed decreased expression in the presence of the TNIP1 risk haplotype (P < 0.001 and P < 0.01, respectively, relative to the nonrisk haplotype); notably, it was found that these genes share a 3-D chromatin network. CONCLUSION Hypomorphic TNIP1 expression results from the combined concordant and opposing effects of multiple risk variants carried on the TNIP1 risk haplotype, with the strongest regulatory effect in B lymphoid lineage cells. Furthermore, the TNIP1 risk haplotype effect extends to neighboring genes within a shared chromatin network.

中文翻译：

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具有相反功能效应的系统性红斑狼疮风险变异作为 TNIP1 和三维染色质网络中其他基因亚形表达的驱动因素的作用。

目的 肿瘤坏死因子诱导蛋白 3 相互作用蛋白 1 (TNIP1) 区域的遗传变异与自身免疫性疾病和 TNIP1 基因表达降低有关。本研究的目的是确定系统性红斑狼疮相关 TNIP1 H1 风险单倍型所导致的驱动 TNIP1 低等态表达的功能遗传机制。方法 使用双荧光素酶表达和电泳迁移率变动分析来评估 11 种风险变异对免疫细胞系模型（EB 病毒 [EBV] 转化的人 B 细胞、Jurkat 细胞和THP-1 细胞），处于静息状态或用佛波醇 12-肉豆蔻酸酯 13-乙酸酯/离子霉素刺激。HiChIP 用于通过检测与 EBV B 细胞中 H3K27ac 标记染色质相关的原位长程 DNA 接触来定义 TNIP1 单倍型的调控 3 维 (3-D) 染色质网络。然后，使用定量逆转录聚合酶链反应 (qRT-PCR) 确定 3-D 染色质网络内基因的表达。结果对 TNIP1 H1 风险单倍型的 50 个单核苷酸多态性进行生物信息学分析，鉴定出 11 个非蛋白质编码变异，很可能影响 TNIP1 基因表达。EBV B 细胞中的 8 个变体、THP-1 细胞中的 5 个变体和 Jurkat 细胞中的 2 个变体对增强子激活表现出各种等位基因效应，导致对 TNIP1 表达的累积抑制效应（风险变体的净效应 -7.14 倍、-6.80 倍，和 -2.44 倍，分别；n > 3)。具体而言，在 EBV B 细胞中，只有 2 个变体（rs10057690 和 rs13180950）表现出增强子活性和核蛋白结合的等位基因特异性丧失（相对于非风险等位基因，每个 P < 0.01）。相反，rs10036748风险等位基因降低了软骨细胞1（bHLHe40/DEC1）中差异表达的转录抑制因子基本螺旋-环-螺旋家族成员40/DEC1（相对于非风险等位基因，P<0.05）和CREB-1（P不EBV B 细胞中显着），导致增强子活性增加（P < 0.05）。HiChIP 和 qRT-PCR 分析显示，TNIP1 单倍型的整体转录抑制延伸至邻近基因 DCTN4 和 GMA2，这两个基因在 TNIP1 风险单倍型存在时也表现出表达降低（分别为 P < 0.001 和 P < 0.01，相对于非风险单倍型）；值得注意的是，我们发现这些基因共享一个 3D 染色质网络。结论 TNIP1 亚形表达是由 TNIP1 风险单倍型上携带的多种风险变异的一致和相反效应联合产生的，其中在 B 淋巴谱系细胞中具有最强的调节作用。此外，TNIP1 风险单倍型效应延伸到共享染色质网络内的邻近基因。