ELLEN ROTHENBERG

..Several reports now reveal how TCF-1 and GATA-3 are mobilized in early T cells and the pathways for their T-lineage specific effects...

..We argue that epigenetic analysis may achieve its greatest impact for illuminating regulatory biology when it is used to locate cis-regulatory elements by catching them in the act of mediating regulatory change...

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..Here we review the current evidence identifying the regulatory components of this commitment pathway, and the first hints of how they work together. Roles for PU.1, GATA-3, and their target genes are highlighted...

..Here, we review insights into T-cell specification and commitment that emerge from a combination of molecular, cellular, and systems biology approaches. The results reveal the regulatory structure underlying this lineage decision...

..We consider the functions of these factors in animals without lymphocytes in terms of discrete program components, which could have been assembled in a new way to create the lymphocyte developmental program approximately 500 My ago...

..1, and Notch-Delta signals, whose counterbalance appears to be essential for T-cell specification...

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..An additional mechanism, still not fully defined, is required just prior to T cell receptor-mediated selection to end plasticity and make T lineage commitment irreversible...

..1, growth factor independence (Gfi)-1, T cell factor (TCF)-1, and Runx factors and their interactions with the Notch pathway to promote T cell development...

..The comparison among them sheds light on the different ways that an essential regulatory input can affect cellular identity...

..However, the response of the population to PU.1 is sharply discontinuous. These studies show a critical role for regulatory context in restricting plasticity, which is probably maintained by interacting transcription factor networks...

..This regulatory mosaic has notable implications for the hierarchy of relationships linking T lymphocytes to other hematopoietic fates...

..Hypothetical models are proposed to indicate the network nodes that are differentially activated in normal T cell lineage progression and in cells diverted to other potential fates...

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..Thus, the SCID thymocytes appear to undergo a normal generation but a premature death as compared with the RAG-2 -/- thymocytes...

..The tissue-specific expression patterns of skate PU.1 and Spi-C suggest that these genes share regulatory as well as structural properties with their mammalian orthologs...

..However, the expression pattern of Vbeta2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles...

..Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells...

..Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts...

..1 versus GATA-1 balance, the intensity of Notch signaling through the CSL pathway, and the ratio of E-box transcription factors to their Id protein antagonists...

..A model is proposed in which discrete changes in functional competence define novel transitions in early thymocyte development, at least some of which may be linked to TCR-beta gene rearrangement before positive or negative selection...

..Therefore we conclude that IL2 gene expression is controlled primarily through a central TH1-specific signaling pathway, which acts through proximal elements, while distal cis-elements exert a secondary modulating effect...

..Our results suggest a close relationship between the pro-T cell and mast cell programs and a previously unknown function for Notch in T lineage fidelity...

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..Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFbeta, and both signals converge to activate the T-cell developmental program...

..Bcl11b was uniquely T lineage restricted and induced by Notch/Delta signaling specifically upon entry into the T lineage differentiation pathway...

..The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment...

..Therefore, HEBAlt and HEBCan are functionally distinct transcription factors, and HEBAlt is specifically required for the efficient generation of early T cell precursors...

..The results also reveal global differences in regulatory alterations triggered by the first T-cell receptor-dependent selection events in fetal and adult thymopoiesis...

..PU.1 expression can downregulate pre-Talpha, Rag-1, and Rag-2 in a dose-dependent manner, and higher PU.1 levels induce Mac-1 and Id-2. Thus, downregulation of PU.1 is specifically required for progression in the T cell lineage...

..The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects...

..We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes...

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..However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals...

..Furthermore, this breakthrough may initiate thymic lymphomagenesis that occurs with high frequency in both NOD-scid and -Rag1null mice...

..The presence of the nuclear matrix targeting sequence is required for Runx-mediated CD4 repression, suggesting that Runx transcription factors are stabilized on the CD4 silencer via association with the nuclear matrix...

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..The adh.2C2 and adh.6D4 clones thus provide an accessible system for defining mechanisms controlling developmental plasticity in early T-cell development...

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..Our results also reveal differences in Notch/Delta dependence at the earliest stages of divergence between developing alphabeta and gammadelta T-lineage cells...

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..1 itself. ..

..Definition of mechanistic requirements for GATA-3 perturbation of T-cell development 3. Attempted rescue of GATA-3 overexpression effects by cotransduction with targets of specific repression. ..

..Definition of mechanistic requirements for GATA-3 perturbation of T-cell development 3. Attempted rescue of GATA-3 overexpression effects by cotransduction with targets of specific repression ..

..This should permit faster and cheaper experiments to reveal how powerful regulatory genes are themselves controlled. ..

..These computational predictions will guide the experimental program and interpret the data it generates. Ellen Rothenberg will lead the experimental component of the project...

..The mechanisms studied here imply that mixed-lineage thymic lymphomas may also result from primary defects of a natural T/myeloid lineage choice. ..

..Specific aim 3: To test the fidelity of T-cell lineage-restricted lL-2 expression by analysis of lL-2/Cre-dependent marking of non-T lineage cells in vivo. ..

..Definition of mechanistic requirements for GATA-3 perturbation of T-cell development 3. Attempted rescue of GATA-3 overexpression effects by cotransduction with targets of specific repression. ..

..This application is to replace the old sorter with a new sorter that will improve service to existing clients and permit the Facility to carry out new flow cytometric applications that have not been available to the user group before. ..

..The mechanisms studied here imply that mixed-lineage thymic lymphomas may also result from primary defects of a natural T/myeloid lineage choice. ..