Thymocyte and thymic epithelial cell (TEC) development are interdependent processes. Although lineage relationships among progressively maturing thymocyte subsets have been characterized, the developmental relationships among TEC subsets are obscure. Because epithelial cells express distinct keratin (K) species as a function of differentiation stage and proliferative status, we used K expression patterns to identify mouse TEC subsets and determine their lineage relationships. CD25ϩ pre-T cells that productively rearrange the TCR␤ locus and express pre-TCR͞CD3 complexes proliferate and differentiate to the CD4 ϩ CD8 ϩ double-positive (DP) stage. The DN to DP transition is accompanied by loss of CD25 expression, prohibition of TCR␤ locus rearrangements, and induction of TCR␣ locus rearrangements. Signaling through the ␣␤TCR͞CD3 complexes on DP thymocytes mediates the positive and negative selection processes that shape the T-cell repertoire. DP thymocytes that are positively selected by self-peptide͞major histocompatibility complex molecules presented on cortical epithelial cells terminate CD8 or CD4 expression and migrate to the thymic medulla.Thymic epithelial cells (TECs) are not only involved in the selection of DP cells, but also promote differentiation of early DN thymocyte precursors. Nude mice that are unable to generate a normal thymic epithelial compartment because of an inactivating whn gene mutation have a primitive thymic anlage devoid of T cells (3). Furthermore, recent studies have shown that the maturation of DN precursors in thymic organ culture requires the presence of major histocompatibility complex class II ϩ TECs (4-6). Although TECs are known to play a critical role in T-cell maturation, the factors that govern TEC development are incompletely understood, particularly in comparison to the well-characterized T-cell developmental process. Nevertheless, it is clear that the establishment of normal thymic architecture and thus, TEC differentiation, depends on thymocyte͞TEC interactions (7,8). This interdependence is apparent in mice that express a human CD3 (hCD3) transgene, which prevents T-cell maturation beyond the primitive CD44 ϩ CD25 Ϫ

Thymic epithelial cells (TECs) in adult mice have been classified into distinct subsets based on keratin expression profiles. To explore the emergence of TEC subsets during ontogeny, we analyzed keratin 8 and keratin 5 expression at several stages of fetal development in normal C57BL/6J mice. In addition, thymic epithelial development and compartmentalization were explored in recombination-activating gene 2/common cytokine receptor γ-chain-deficient and Ikaros-null mice that sustain early and profound blocks in thymocyte differentiation. The results demonstrate that initial patterning of the thymic epithelial compartment as defined by differential keratin expression does not depend on inductive signals from hematopoietic cells. However, thymocyte-derived signals are required during late fetal stages for continued development and maintenance of TEC subsets in the neonate and adult.

The coactivator-associated arginine methyltransferase, CARM1, is a positive regulator of transcription. Using high density protein arrays, we have previously identified in vitro substrates for CARM1. One of these substrates, TARPP (thymocyte cyclic AMP-regulated phosphoprotein), is expressed specifically in immature thymocytes. Here, we have demonstrated that TARPP is arginine-methylated at a single residue, Arg 650 , both in vitro and in vivo. In addition, recombinant TARPP is not methylated by extracts from Carm1 ؊/؊ cells, indicating that there is no redundancy in this pathway. We show that thymi from Carm1 ؊/؊ embryos (E18.5) have a 5-10-fold reduction in cellularity compared with wild type littermates. Flow cytometric analysis of thymocytes revealed a decrease in the relative proportion of double negative thymocytes in Carm1 ؊/؊ embryos because of a partial developmental arrest in the earliest thymocyte progenitor subset. These results demonstrate that CARM1 plays a significant role in promoting the differentiation of early thymocyte progenitors, possibly through its direct action on TARPP.

CARM1 is one of nine protein arginine methyltransferases that methylate arginine residues in proteins. CARM1 is recruited by many different transcription factors as a positive regulator. Gene targeting of CARM1 in mice has been performed, and knock-out mice, which are smaller than their wild-type littermates, die just after birth. It has been proposed that CARM1 has functions that are independent of its enzymatic activity. Indeed, CARM1 is found to interact with a number of proteins and may have a scaffolding function in this context. However, CARM1 methylates histone H3, PABP1, AIB1, and a number of splicing factors, which strongly suggests that its impact on transcription and splicing is primarily through its ability to modify these substrates. To unequivocally establish the importance of CARM1 enzymatic activity in vivo, we generated an enzyme-dead knock-in of this protein arginine methyltransferase. We determined that knock-in cells and mice have defects similar to those seen in their knock-out counterparts with respect to the time of embryo lethality, T cell development, adipocyte differentiation, and transcriptional coactivator activity. CARM1 requires its enzymatic activity for all of its known cellular functions. Thus, small molecule inhibitors of CARM1 will incapacitate all of the enzyme's cellular functions.