A Novel Gene Essential for the Development of Single Positive Thymocytes

Abstract

A critical step during intrathymic T-cell development is the transition of CD4؉ CD8 ؉ double-positive (DP) cells to the major histocompatibility complex class I (MHC-I)-restricted CD4؊ CD8 ؉ and MHC-II-restricted CD4 ؉ CD8 ؊ single-positive (SP) cell stage. Here, we identify a novel gene that is essential for this process. Through the T-cell phenotype-based screening of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we established a mouse line in which numbers of CD4 and CD8 SP thymocytes as well as peripheral CD4 and CD8 T cells were dramatically reduced. Using linkage analysis and DNA sequencing, we identified a missense point mutation in a gene, E430004N04Rik (also known as themis), that does not belong to any known gene family. This orphan gene is expressed specifically in DP and SP thymocytes and peripheral T cells, whereas in mutant thymocytes the levels of protein encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice, and their phenotype was virtually identical to that of the ENU mutant mice, thereby confirming that this gene is essential for the development of SP thymocytes.The differentiation step from the double-positive (DP) to single-positive (SP) thymocyte stage is critically regulated by signals originating from the T-cell receptor ␣/␤ (TCR␣/␤) expressed on their surface (3,5,16,17). By using reverse genetic approaches by knocking out or overexpressing various genes that are expected to be involved in TCR signaling, including its ligand major histocompatibility complex molecules and coreceptors CD4 and CD8, the roles of these genes in T-cell development have been investigated intensively (11,12). However, to identify totally unknown mechanisms in T-cell development, the forward genetic approach is required. Nethyl-N-nitrosourea (ENU) is a potent mutagen that randomly induces point mutations throughout the genome in a dosedependent manner, and ENU mutagenesis has been a representative forward genetic strategy (4, 15). We have been screening phenotypes of ENU-mutagenized mice, focusing on defects in T-cell development. MATERIALS AND METHODSMice. C57BL/6 (B6) and B6Ly5.1 congenic mice were purchased from CLEA Japan, Inc. All mice were maintained in the animal facility at the RIKEN Research Center for Allergy and Immunology, and all experiments were done in accordance with institutional guidelines for animal care. ENU mutagenesis. ENU was administered to male C57BL/6J mice, and their sperm was mated to wild-type eggs and preserved as founder embryos (8, 21) for use in our screening.Mapping and sequencing. Phenodeviants that showed CD3 ϩ cell reduction were crossed to wild-type C3H/HeJ mice to test for phenotype transmission and for the genetic mapping of the causative genes. Single-nucleotide polymorphism (SNP) mapping was performed as described elsewhere (13). For sequencing, we focused on the ptprk and E430004N04Rik genes, which were among candidate genes listed by PosMed (21) (http://omicspace.riken.jp). cDNA and genomic DNA were amplified by PCR and sequenced us...

Gasp, a Grb2-associating protein, is critical for positive selection of thymocytes

“…We would like to note that, during the review process of this article, several other groups' reports describing the same gene under the name "Themis" were published (33)(34)(35)40).…”

Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability

“…Despite the absence of strong biochemical evidence, it was presumed that Themis1 likely functions as a positive regulator of TCR signaling because several defects in Themis1 −/− mice, such as a marked impairment of both positive and negative selection, were consistent with a deficiency in TCR signaling (2)(3)(4)(5)(6). It was subsequently shown that Themis1 interacts with Vav1 and that Vav1 phosphorylation is reduced in Themis1 −/− thymocytes, results that also implied a positive role for Themis1 in TCR signaling (9).…”

“…We and others identified the previously uncharacterized protein Themis1 as being required for the positive selection and maturation of T cells in the thymus (2)(3)(4)(5)(6). Themis1 lacks any discernable catalytic domain, but it contains two previously uncharacterized cysteine-based globular domains (named "CABIT") of unknown structure and function (3).…”

An Epistatic Interaction between Themis1 and Vav1 Modulates Regulatory T Cell Function and Inflammatory Bowel Disease Development

“…The ablation of Themis1 in C57BL/6 mice results in impaired T cell development, particularly of the CD4 + lineage (17)(18)(19)(20)(21). After TCR engagement, Themis1 is phosphorylated by Lck and Zap-70, and recruited to the transmembrane adaptor protein LAT (22,23).…”