Overexpression of the Helix–Loop–Helix protein Id2 blocks T cell development at multiple stages
Introduction
T cell differentiation and selection in the thymus is an ideal model system for studying mammalian development. Committed T cell precursors that first arrive in the thymus from the bone marrow express low levels of CD4, but quickly downregulate it to become CD4−CD8− double-negative (DN) cells (Wu et al., 1991). The DN population can be further divided into distinct developmental stages based on the surface expression of CD25 and CD44 (Godfrey et al., 1993). The earliest thymocyte is CD44loCD25−, but rapidly upregulates expression of CD44 and subsequently CD25, to become the CD44+CD25+ DN population. These cells express the RAG genes and begin to rearrange the β chain genes of the T-cell Antigen Receptor (TCR) (Godfrey et al., 1993, Mombaerts et al., 1992b, Shinkai et al., 1993, Shinkai et al., 1992). During the rearrangement process, the DN thymocyte subsequently downregulates expression of CD44 and CD25 (Mombaerts et al., 1992b, Shinkai et al., 1993, Shinkai et al., 1992) and upregulate CD8 to become the CD4−CD8+TCR− population. This subclass is one of the most rapidly proliferating populations in the thymus. Should the β chain gene rearrangement be successful, the thymocyte upregulates both CD4 and CD8 (Mombaerts et al., 1992a, Mombaerts et al., 1992b, Shinkai et al., 1993, Shinkai et al., 1992). The resulting CD4+CD8+ or double positive (DP) population undergoes several important developmental processes. First, the DP cell rearranges the TCR α chain gene (Levelt et al., 1995, Petrie et al., 1993). Should this rearrangement also prove successful, the thymocyte undergoes the TCR-mediated selection processes that insure properly restricted mature T cell populations (Bevan et al., 1994). Survivors then downregulate either CD4 or CD8, leading to the mature single-positive populations (Kaye et al., 1989, Sha et al., 1988a, Sha et al., 1988b). This downregulation is considered to be an important event in the final maturation of the T cell, and, for the CD4 gene, is due primarily to the action of a subclass-specific silencer (Duncan et al., 1996, Sawada et al., 1994, Siu et al., 1994).
The basic–Helix–Loop–Helix (bHLH) protein family has been implicated in the control of cellular differentiation and growth (Kadesch, 1992, Murre et al., 1994). These proteins are transcription factors that contain a common protein–protein interaction domain referred to as the helix–loop–helix region that allows these proteins to dimerize and a DNA-binding domain composed of basic amino acids (Baer, 1993, Henthorn et al., 1990, Hu et al., 1992, Lassar et al., 1991, Murre et al., 1989, Weintraub et al., 1991a, Weintraub et al., 1991b, Weintraub et al., 1989). One subclass, the Id proteins, contains only the HLH protein–protein interaction domain, which enables them to dimerize with other HLH proteins both in vivo and in vitro (Benezra et al., 1990, Jen et al., 1992, Kreider et al., 1992, Sun et al., 1991, Wilson et al., 1991). Because the Id proteins lack a basic region, they inhibit the function of the bHLH proteins to which they bind by abrogating their ability to bind DNA. The inhibition of bHLH transcription factor function by Id proteins has many critical developmental consequences. For example, the overexpression of Id inhibits factor-dependent differentiation of immature cells (Jen et al., 1992, Kreider et al., 1992), whereas the inhibition of Id expression leads to uncontrolled proliferation (Barone et al., 1994, Peverali et al., 1994). In vivo, Id proteins are highly expressed in early development and are downregulated in terminally-differentiated cells, consistent with the hypothesis that these proteins play a role in differentiation and proliferation (Benezra et al., 1990, Biggs et al., 1992, Kawaguchi et al., 1992).
Recent studies have suggested a role for bHLH proteins in T cell development and function. The bHLH proteins E12, E47, HEB, and the Id HLH proteins are all expressed in the thymus, consistent with a role for these proteins in T cell development (Hu et al., 1992, Riechmann et al., 1994, Roberts et al., 1993). Mice with targeted disruption of either the HEB or E2A gene have a T-cell developmental block at an early CD4−CD8− stage and eventually succumb to aggressive thymic lymphomas, indicating that the bHLH proteins HEB, E12 and E47 play an important role in T cell development and lymphomagenesis (Bain et al., 1997, Zhuang et al., 1996). Interestingly, although mice heterozygous for targeted disruptions either in the HEB or the E2A genes are normal, mice of the +/−HEB +/−E2A genotype have an early T cell developmental block at the same stage as the homozygous HEB or E2A null mice (Zhuang et al., 1996). These observations support the hypothesis that the balance of all bHLH proteins controls normal thymopoiesis rather than any one specific bHLH protein. However, overexpression of the Id1 gene in lymphocytes significantly alters B cell but not T cell development (Sun, 1994). Since this Id protein can bind to both HEB and E2A gene products, in principle the overexpressed Id1 should decrease significantly the levels of functional E2A and HEB, and thus according to this hypothesis the Id1 transgenic mice would have similar T cell developmental blocks as the homozygous E2A and HEB null mice.
To study the role of the bHLH proteins in T cell development, we generated transgenic mice that overexpress a different Id protein, Id2, in T cells. We have determined that the Id2 transgenic mice have an early stage-specific block in T cell development at a stage similar to the homozygous E2A and HEB mice and the double-heterozygote null E2A and HEB mice. Progeny mice from most but not all transgenic founder lines develop aggressive T cell hyperproliferation as they age. Interestingly, although this hyperproliferation resembles the growth of transformed cells, these cells are not monoclonal, suggesting that the expanded T cell populations in the tumorous Id2 transgenic mice are the result of multiple independent transformation events. We thus conclude that Id2 plays an important role in proliferation and differentiation early in thymopoiesis and predisposes T cells to oncogenic transformation.
Section snippets
Generation of transgenic mice
A 672bp restriction enzyme fragment containing the coding region of Id2 was cloned into the BamHI site of the p1017 vector (Wu et al., 1991). This vector contains the lck proximal promoter, which induces expression at high levels in the thymus and low levels in the peripheral T cells. Transgenic mice were generated with this construct using previously published protocols (Sawada et al., 1994). In brief, construct DNA was excised from the vectors and purified on a 10–40% continuous sucrose
Alterations in T cell subclasses in Id2 transgenic mice
To determine the role of Id2 in T cell development, we generated a transgenic construct containing the Id2 gene under the transcriptional control of the lck promoter (Abraham et al., 1991). This promoter construct expresses the cloned cDNA at high levels early in T cell development and lower but detectable levels in mature T cells. We generated and established six independent founder lines and analyzed 8–12 week-old progeny from each for alterations in lymphocyte development. To assess levels
The role of Helix–Loop–Helix proteins in T cell development
Our data are consistent with the hypothesis that the bHLH protein family plays an important role at several stages early in thymic development. Although the precise roles of each of the bHLH proteins in thymopoiesis remains to be elucidated, studies suggest that HEB, E12/E47, and Id2 each play distinct roles. These factors may either play a specific role in developing thymocytes by regulating transcription of T cell specific genes, or they may play a more general role by maintaining a delicate
Acknowledgements
We thank Julia Lindenberg and Daniel Ng for assistance, Dr Roger Perlmutter for the p1017 lck construct, Drs Andrew Henderson, Stephen Brunnert, and Frank Costantini for their advice, and Dr Chris Schindler for critical evaluation of the manuscript. This work was supported by grants from the American Cancer Society (RPG98-185-01-CIM) and the Irma T. Hirschl-Monique Weill Caulier Charitable Trust to GS and from the New York City Chapter of the Arthritis Foundation (CU51177701) to EWM.
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