No abstract

Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we have utilized Rag1−/− blastocyst complementation and in vitro embryonic stem (ES) cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag 1−/− chimeras displayed a dramatic reduction in thymic cellularity compared to control mice. Using ESC differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag 1−/− chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared to controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent early-stage, and distinct, Beclin 1-independent, late stage processes.

Locus control regions (LCRs) are thought to provide a dominant tissue‐specific open chromatin domain that allows for proper gene regulation by enhancers/silencers and their associated transcription factors. Expression of the T‐cell receptor alpha (TCRα) gene is limited to T cells and its locus exists in different chromatin configurations in expressing and non‐expressing cell types. Here we show that eight DNase I‐hypersensitive sites in the TCRα locus comprise an LCR that confers T‐cell compartment‐specific expression upon a linked heterologous transgene. Removal of the three 5′‐most hypersensitive sites of this LCR, containing TCRα enhancers/silencers, abolishes tissue‐differential chromatin structure and results in transgene expression in all tissues examined. The remaining five DNase I‐hypersensitive sites therefore constitute a novel control element possessing a widely active chromatin‐opening function that allows for ubiquitous expression of a linked transgene in all transgenic founder mice. Furthermore, these data show that cis‐acting elements without inherent LCR activity can dominantly modulate chromatin structure to determine tissue‐specific gene expression in vivo.