Mitf regulates gene expression networks implicated in B cell homeostasis, germinal center responses, and tolerance. Mitf regulates gene expression networks implicated in B cell homeostasis, germinal center responses, and tolerance

The microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure. Two complementary mouse models of Mitf and MiT deficiency – the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B) – were used to identify MiT family candidate target genes and pathways. Both models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation implicating non-autonomous B-lymphocyte mechanisms caused by systemic absence of Mitf. These studies clarify Mitf's role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. Overall design: To investigate the B cell-specific function of Mitf in comparison to other MiT family members, we employed two mouse models: TDN-B, expressing a dominant negative MiT inhibitor solely in B cells, and a germline recessive null Mitf mutation, Mitfmi-vga9/mi-vga9, which does not inhibit other MiT family proteins. To distinguish between B cell abnormalities due to Mitf inactivation and those influenced by T cell function, we utilized TDN transgenic mice (TDN-B/T). These mice express TDN in both B and T cells, leading to impaired T cell function. Subsequently, we performed RNAseq analysis on resting B cells from the different MiT/Mitf-impaired strains mentioned above. This analysis aimed to identify differentially regulated genes that might elucidate underlying mechanisms explaining the common and unique B cell abnormalities observed in each model.

小眼畸形转录因子（microphthalmia transcription factor, Mitf）已被证实可调控B细胞活化与免疫耐受。然而，其发挥调控作用的B细胞特异性机制，以及区分Mitf与其他高度同源的Mitf/TFE（MiT）转录因子（包括Tfe3、Tfeb和Tfec）的机制，仍有待阐明。本研究采用两种互补的Mitf与MiT缺陷小鼠模型——分别为Mitfmi-vga9/mi-vga9系统性功能丧失突变模型，以及通过转基因表达显性负效（trans-dominant negative, TDN）蛋白实现B细胞特异性MiT家族失活的TDN-B模型——以鉴定MiT家族的候选靶基因与信号通路。两种模型均出现自发性脾肿大，伴随浆细胞数量增多、自身抗体滴度升高与蛋白尿。上述异常表现依赖于辅助T细胞的存在，但与系统性Mitf失活所引发的其他非B细胞固有效应无关。在C57BL/6-Faslpr/lpr遗传背景下，B细胞中MiT的失活会加重狼疮样自身免疫病的相关表型。在两种模型中，对离体静息B细胞开展RNA测序（RNAseq）分析结果显示，调控细胞周期、生发中心反应及浆细胞分化的基因转录水平显著上调。在两种模型中均被显著上调的基因包括Socs6、Isp53（Baiap1）、S1pR2及IgG2b/c。Mitf敲除的B细胞（而非TDN-B细胞）呈现出I型干扰素调控异常的特征，这提示系统性Mitf缺失会引发非自主性B淋巴细胞机制异常。本研究明确了Mitf的双重功能：1）作为B细胞固有生发中心程序的关键调控因子，通过全新靶基因影响自身免疫耐受；2）作为系统性炎症过程的调控因子，可作用于B细胞微环境。整体实验设计：为探究Mitf相较于其他MiT家族成员的B细胞特异性功能，本研究采用了两种小鼠模型：仅在B细胞中表达显性负效MiT抑制剂的TDN-B模型，以及不抑制其他MiT家族蛋白的生殖系隐性纯合Mitf敲除突变模型Mitfmi-vga9/mi-vga9。为区分由Mitf失活导致的B细胞异常与受T细胞功能影响的异常，我们使用了同时在B细胞和T细胞中表达TDN的转基因小鼠（TDN-B/T），该模型的T细胞功能存在缺陷。随后，我们对上述不同MiT/Mitf功能受损品系的静息B细胞进行了RNAseq分析，旨在鉴定差异表达基因，以阐明解释各模型中观察到的共通与独特B细胞异常的潜在机制。