Abstract:
Thymocyte development is a complex process that requires precise regulation of
differentiation and proliferation. Basic helix-loop-helix (bHLH) transcription factors
have been shown to be crucial for proper T cell development. HEB and E2A are
structurally and functionally related E proteins of the bHLH family. These proteins
directly regulate the expression of a number of genes essential for lymphocyte
development in a lineage- and stage-specific manner. Abrogation or compromise of their
function results in the manifestation of B and T cell developmental defects.
Genetic and biochemical studies have provided evidence of a significant degree of
functional redundancy among E proteins. The existence of compensational abilities
among different E proteins has hampered the investigation and elucidation of E protein
function. As such, single gene knockouts demonstrate only limited defects in lymphocyte
development. Double E2A-HEB knockouts that could eliminate E protein redundancy
are embryonic lethal. In addition, conventional gene knockouts are not well-suited for
discerning between intrinsic and extrinsic defects caused by E protein disruption.
To eliminate functional compensation and to test the T cell intrinsic roles of E
proteins during thymocyte development, we developed a conditional HEB-E2A double
knockout. Specifically, we employed a loxP/Lck-Cre recombinase system to drive E
protein deletion during early thymocyte development. Using this approach, we were able
to reveal overlapping roles for HEB and E2A in thymocyte development that had been
obscured in previous single gene knockout studies.
We find that simultaneous deletion of HEB and E2A results in a severe block in
thymocyte development at the DN to DP stage transition. This developmental block is
accompanied by a dramatic decrease in total thymic cellularity, an increase in apoptosis,
and a reduction of pTα expression. These developmentally arrested thymocytes exhibit
increased proliferation in vivo and dramatic expansion ex vivo in response to IL-7
signaling. Our findings suggest that E2A and HEB are not only critical for the regulation
of T cell differentiation but are also necessary to retain developing thymocytes in cell
cycle arrest prior to pre-TCR expression. Together, these results imply that E proteins
are required to coordinate thymocyte differentiation and proliferation.
