Early Growth Response Gene 3 Regulates Thymocyte Proliferation during the Transition from CD4−CD8− to CD4+CD8+1

Abstract

In thymocytes developing in the αβ lineage, the transition from CD4, CD8 double negative (DN) to CD4, CD8 double positive (DP) is associated with several rounds of cell division and changes in the expression of multiple genes. This transition is induced by the formation of a pre-TCR that includes a rearranged TCR β-chain and the pre-TCR α-chain. The mechanism by which the pre-TCR influences both gene expression and proliferation has not been defined. We have evaluated the role played by early growth response gene 3 (Egr3) in translating pre-TCR signals into differentiation and proliferation. Egr3 is a transcriptional regulator that contains a zinc-finger DNA binding domain. We find that Egr3-deficient mice have a reduced number of thymocytes compared with wild-type mice, and that this is due to poor proliferation during the DN to DP transition. Treatment of both Egr3+/+ and Egr3−/− mice on the Rag1−/− background with anti-CD3ε Ab in vivo results in similar differentiation events, but reduced cell recovery in the Egr3−/− mice. We have also generated transgenic mice that express high levels of Egr3 constitutively, and when these mice are bred onto a Rag1−/− background they exhibit increased proliferation in the absence of stimulation and have pre-TCR α-chain and CD25 down-regulation, as well as increased Cα expression. The results show that Egr3 is an important regulator of proliferation in response to pre-TCR signals, and that it also may regulate some specific aspects of differentiation.

Opposing regulation of T cell function by Egr‐1/NAB2 and Egr‐2/Egr‐3

“…We also took advantage of an Egr-3-overexpressing transgenic (Egr-3 TG) mouse. This mouse has previously been described as having lower percentages of thymic CD4 + T cells and increased percentages of thymic CD8 + and CD4 -CD8 -T cells [26]. Peripheral blood analysis of Egr-3 TG mice indicated lower CD4 + T cell percentages than in WT controls; however, 6.5 + CD4 + T cell percentages were not significantly different (data not shown).…”

“…In contrast, an inhibitor of NF-AT/AP-1 association could potentially promote a tolerogenic program, in part, by leaving TCR-induced expression of Egr-2 and Egr-3 intact. [25,26]. The Egr3 -/-mice and Egr3 TG mice were backcrossed onto the B10.D2 6.5 + TCR-transgenic background for at least nine and five generations, respectively.…”

Enforced Expression of Spi-B Reverses T Lineage Commitment and Blocks β-Selection

“…Importantly, although induction of Egr1 appeared normal in Spi-B-expressing cells, induction of Egr2 and Egr3 was impaired, as was the induction of the Egr protein target, Id3 (57). Although we have previously determined that induction of the Egr proteins is sufficient for traversal of the ␤-selection checkpoint, it should be noted that ablation of Egr3 alone partially impairs development beyond the ␤-selection checkpoint (34). Moreover, Egr2 and Egr3 are much more potent inducers of the helix-loop-helix factor, Id3, than is Egr1 (data not shown) (32).…”

“…We and others have demonstrated that the Egr family of Zn 2ϩ finger transcription factors plays an important role in traversal of the ␤-selection checkpoint (32)(33)(34). To determine whether induction of the Egr proteins were perturbed by Spi-B overexpression, we performed RT-PCR Southern blots on TAC:CD3⑀ stimulated Scid.adh cells transduced with vector alone (LZRS) or with Spi-B (LZRS-Spi-B).…”

“…Ϫ ; DP, CD4 ϩ CD8 ϩ ; DC, dendritic cell; Egr, early growth response; TAC:CD3⑀, human IL-2R␣ exo and transmembrane domains fused to the cytoplasmic domain of CD3⑀; IRES, internal ribosomal entry site; eGFP, enhanced GFP; CFP, cyan fluorescent protein; LZRS, LZRSpBMN-linker-IRES-eGFP; pMIC, p-MSCV-IRES-CFP; FTOC, fetal thymic organ culture; SCF, stem cell factor; SA, streptavidin; MUG, methyl-umbelliferyl-␤-D-galactoside; MHC-II, MHC class II; IRF, IFN regulatory factor; Id3, inhibitor of DNA binding 3. signaling depends heavily on the activities of the lck and ZAP-70 tyrosine kinases (20 -23), the molecular adaptors Src homology 2 domain-containing leukocyte protein, 76 kDa, and linker for activation of T cells (24 -26), as well as a number of DNA binding proteins including high mobility group box factors, Tcf-1 and Lef-1 and their coactivator ␤-catenin (27,28), E box family basic helix-loop-helix proteins (29,30), c-myb (31), and the early growth response (Egr) family of zinc finger transcription factors (32)(33)(34). Despite the identification of a number of effectors of pre-TCR signaling, our understanding of how the ␤-selection differentiation program is elaborated in response to pre-TCR signals remains incomplete.…”