Whereas there is now ample evidence that the E2A proteins activate the expression of a large subset of target genes, including Ptcra, Hes1, Notch1, Notch3, Cd3e, Tle3, Tle5 and Tcrb, much less is known as to how the genome-wide activity of E2A is modulated. It is generally assumed that the DNA binding activity of E2A is antagonized by the induction of Id3 expression mediated by pre-TCR signalling. However, this has remained to be proven. Here we demonstrate at a global scale that E2A occupancy decreases at a large subset of putative enhancer elements. Specifically, we found that 692 bound regions were restricted to the DN3 compartments, 247 sites overlapped between the DN3 and DN4 compartments and 93 E2A bound sites were detected in the DN4 compartment. The decline in the number of E2A-bound sites at the pre-TCR checkpoint is striking and consistent with an increase in Id3-GFP expression in DN3b. Thus, upon pre-TCR mediated signaling, Id3 abundance is increased to suppress E2A occupancy at the vast majority of binding sites. We suggest that this decrease in global occupancy underpins the mechanism that promotes β-selection.
The global studies showed that the consensus DNA sequence (CAGCTG) associated with E2A occupancy is identical in two entirely different cell lineages: DN3 and pro-B cells30
. Thus, E2A proteins recognize the same binding site in committed B- and T-cells. However, the enhancer repertoires associated with E2A binding in B versus T cells are quite distinct. E2A binding to cistromic elements is highly enriched for EBF binding sites in pro-B cells whereas DN3 cells do not show such an association. Similarly, whereas E2A binding sites are frequently associated SCL/TAL consensus-binding sites in DN3 cells, E2A bound-sites are not enriched for SCL/TAL consensus sequences in pro-B cells. Thus, whereas E2A binds to the same consensus-binding site in the B- versus T-cell lineage, E2A occupancy is associated with different enhancer repertoires in pro-B versus DN3 cells. These data indicate that it is the difference in collaborative interactions at cis-regulatory elements that underpin the mechanism by which E2A proteins promote specific programs of gene expression in distinct lineages.
The cistromic elements associated with E2A occupancy in DN3 cells are frequently enriched for RUNX as well as ETS consensus binding sites. RUNX1 binds in the DN3 compartment to sites present in the CD4 silencer and Ets-1 has been demonstrated to play an essential role in β-selection as well as allelic exclusion41,42
. The collaborative interactions involving E2A and RUNX are of particular interest. Thus, the global analysis confirmed previous observations, identified novel targets, new participants as well as novel links between participants and showed how pre-TCR mediated signaling, at a global and mechanistic scale, promotes developmental progression.
The data also provided new and surprising roles for Id3 in maintaining the naïve CD4 T cell fate. TFH
cells are not normal residents of the thymus. Thus, we are now faced with the question as to how Id3 ablation results in the development of TFH
-like cells in the thymus? The data indicate that a subset of genes associated with at TFH
-like program of gene expression, including CXCR5, is directly regulated by the E2A versus Id3 ratio. However, since only a fraction of CD4SP thymocytes express CXCR5, it seems unlikely that direct regulation by E-proteins is the only mechanism that underpins the aberrant development of TFH
-like cells in Id3-/-
thymi. Rather, we suggest that the development of abnormal effector/memory cells in Id3-ablated thymocytes is, in part, a consequence of compromised TCR mediated signaling. It is now well established that Id3 acts down-stream of TCR mediated signaling18
. Id3-deficient thymocytes show severe defects in positive selection in transgenic mouse models and develop autoimmune disease21,44
. Thus the absence of Id3 may alter the threshold to thymic selection, resulting in the positive selection of cells that normally would be negatively selected, contributing to the aberrant activation of genes associated with an effector/memory phenotype. Of interest with regard to the defect in positive selection in Id3–/–
mice is the observation that the E2A/Id3 ratio regulates the expression of Zap70. Recent observations have indicated that altered thymic selection caused by a mutation in the Zap70 locus leads to the development of autoimmune disease43
. It is conceivable that altered levels of components involved in TCR mediated signal strength such as ZAP-70 contribute to the defects in positive selection observed in Id3–/–
mice as well as the aberrant development of effector and/or memory-like cells. Consistent with this model are recent findings indicating that positive selection is highly sensitive to the dosage of ZAP-70 expression 45
. External factors may contribute as well to the aberrant development of TFH
-like cells in Id3–/–
thymocytes, as observed for the development of innate CD8-like cells27
. In sum, we suggest that the aberrant activation of gene expression associated with an effector/memory phenotype in Id3-/-
thymocytes is a result of aberrant E-protein activity, compromised TCR signaling and an abnormal thymic environment.
Do these data suggest that E- and Id-proteins modulate the development of follicular B and T cells in the peripheral organs? Previous observations have indicated a decrease in the number of naïve T cells in Id3-/-
. We have confirmed these findings but suggest that because of the lymphopenic environment in Id3-/-
mice the defect in the naïve peripheral compartments may be caused, at least in part, by homeostatic proliferation. E2A+/-
mice show a substantial defect in follicular B cell compartments whereas Id3-deficient mice show an increase in the number of follicular B cells46
. Thus, the E2A/Id3 and Bcl6/CXCR5 connection might be shared between follicular B and T cells. Finally, we note that it remains to be determined as to how closely related Id3-/-
-like cells observed in the spleen are to wild-type TFH
cells and it will be essential to ablate Id3, E2A as well as HEB in peripheral CD4 cells in order to carefully evaluate their roles in peripheral TFH
We have previously demonstrated that Id3 expression in DP thymocytes is directly regulated by TCR mediated signaling18
. Consistent with these data we show that Id3 expression progressively increases beyond the pre-TCR and the TCR checkpoints and remains high in naïve peripheral T cells. However, Id3 expression in the effector/memory compartment is quite distinct. It shows a bimodal pattern of expression including a substantial fraction of the effector/memory compartments exhibiting barely detectable levels of Id3 expression. How is Id3 expression differentially regulated? We would like to suggest that it is the difference in signal strength mediated by the TCR that determines Id3 abundance. Thus, upon recognition of self-antigens in the thymus, Id3 expression becomes elevated as described previously18
. We speculate that upon exposure to non-self antigens in the peripheral organs Id3 expression is modulated again to promote differentiation, into either the effector and/or memory cell lineages. Although still to be proven, we propose that it is the difference in Id3 abundance in the naïve versus effector and memory compartments that ultimately determines peripheral T cell fate.