Faculty Bibliography

Bloom's syndrome is a genetic disorder characterized by increased incidence of cancer and an immunodeficiency of unknown origin. The BLM gene mutated in Bloom's syndrome encodes a DNA helicase involved in the maintenance of genomic integrity. To explore the role of BLM in the immune system, we ablated murine Blm in the T-cell lineage. In the absence of Blm, thymocytes were severely reduced in numbers and displayed a developmental block at the beta-selection checkpoint that was partially p53 dependent. Blm-deficient thymocytes rearranged their T-cell receptor (TCR) beta genes normally yet failed to survive and proliferate in response to pre-TCR signaling. Furthermore, peripheral T cells were reduced in numbers, manifested defective homeostatic and TCR-induced proliferation, and produced extensive chromosomal damage. Finally, CD4(+) and CD8(+) T-cell responses were impaired upon antigen challenge. Thus, by ensuring genomic stability, Blm serves a vital role for development, maintenance, and function of T lymphocytes, suggesting a basis for the immune deficiency in Bloom's syndrome.

The developmental origin of type I interferon (IFN)-producing plasmacytoid dendritic cells (PDCs) is controversial. In particular, the rearrangement of immunoglobulin heavy chain (IgH) genes in murine PDCs and the expression of pre-T cell receptor alpha (pTalpha) gene by human PDCs were proposed as evidence for their "lymphoid" origin. Here we demonstrate that PDCs capable of IFN production develop efficiently from both myeloid- and lymphoid-committed progenitors. Rearranged IgH genes as well as RAG transcripts were found in both myeloid- and lymphoid-derived PDCs. The human pTalpha transgenic reporter was activated in both myeloid- and lymphoid-derived PDCs at a level comparable to pre-T cells. PDCs were the only cell population that activated murine RAG1 knockin and human pTalpha transgenic reporters outside the lymphoid lineage. These results highlight a unique developmental program of PDCs that distinguishes them from other cell types including conventional dendritic cells.

Using a human CD25 reporter transgene controlled by regulatory sequences from the gene encoding pre-T cell receptor alpha, we identified a common lymphocyte precursor (CLP-2) population that, in contrast to the previously identified CLP-1 population, was c-Kit-B220+. In short-term culture, the CLP-2 could be derived from the CLP-1 subset, and contained cells that in clonogenic assays were assessed to be bipotent precursors of T and B cells. Intravenous injection of bone marrow cells yielded a selective accumulation of CLP-2 thymic immigrants that in thymic organ culture generated mature alphabeta T cells. Although the CLP-2 subset may represent the most differentiated population with T cell potential before commitment to the B cell lineage, other subsets of thymic immigrants capable of generating T cells may exist

We demonstrate here that "promiscuous" expression of myeloid or lymphoid genes precedes lineage commitment in hematopoiesis. Prospectively purified single common myeloid progenitors (CMPs) coexpress myelo-erythroid but not lymphoid genes, whereas single common lymphoid progenitors (CLPs) coexpress T and B lymphoid but not myeloid genes. Genes unrelated to the adopted lineage are downregulated in bipotent and monopotent descendants of CMPs and CLPs. Promiscuous gene expression does not alter the biological potential of multipotent progenitors: CMPs with an activated endogenous M lysozyme locus yield normal proportions of myelo-erythroid colonies, and CLPs expressing the pre-T cell receptor alpha gene differentiate into normal numbers of B cells. Thus, the accessibility for multiple myeloid or lymphoid programs promiscuously may allow flexibility in fate commitments at these multipotent stages.

A transgenic reporter mouse strain, which expressed the human CD25 (hCD25) surface marker as a reporter under the control of the pre-T cell receptor alpha(pTalpha) promoter, was used to identify lymphoid precursors that expressed pTalpha intracellularly. The hCD25 reporter marked intra- and extrathymic precursors of lymphocytes but not myeloid cells. The earliest intrathymic precursors were CD4(lo)CD8(-)CD25(-)CD44(+)c-Kit(+) cells that expressed elevated levels of Notch-1 mRNA. Clonogenic assays showed that the extrathymic precursors were common lymphoid progenitors (CLPs) that included CD19(-), B220(+), Thy1(+) and CD4(+) cells. Thus, the pTalpha reporter can be used to trace lymphopoiesis between CLPs and alphabeta T cells. The slower extinction of the hCD25 reporter compared to pTalpha enabled us to define points at which pTalpha(-) lineages branched off

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of the pre-T cell receptor alpha (pTa) gene, a T-cell-specific transcriptional target of Notch. The pTa enhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway.

The expression of the pre-T cell receptor alpha (pTa) gene occurs exclusively in immature T lymphocytes and is regulated by poorly defined mechanisms. We have analyzed the role of the upstream enhancer in pTa expression using conventional and bacterial artificial chromosome (BAC) reporter transgenes. The deletion of the enhancer completely abolished the expression of pTa BAC reporter in transgenic mice. Conversely, the combination of pTa enhancer and promoter targeted transgenes specifically to immature thymocytes, recapitulating the expression pattern of pTa. The core enhancer is conserved between mice and humans and contains a critical binding site for the transcription factor c-Myb. We also show that pTa promoter contains a conserved tandem E box site activated by E protein, HEB. These data establish the enhancer as a critical element regulating pTa gene expression and identify additional targets for c-Myb and E proteins in T cell development.

We recently characterized a genomic region located upstream of the mouse pre-T cell receptor alpha (pTa) gene, which controls pTa expression in pre-T cells. We now report an unexpected homology between this region and a region in the mouse X chromosome inactivation center between the 3' end of the Xist gene and the start of an antisense transcript Tsix. The homology is extended over 4 kb of genomic sequence split by an expanded repeat region and is observed only in the mouse, not in the rat. Despite high sequence similarity to the pTa transcriptional enhancer, the homologous X chromosome fragment appears to have lost its enhancer activity. These data underscore the complex organization of the mouse genome and, in particular, of the X chromosome inactivation center.

The pre-T cell receptor alpha (pTalpha) protein is a critical component of the pre-T cell receptor complex in early thymocytes. The expression of the pTalpha gene is one of the earliest markers of the T cell lineage and occurs exclusively in pre-T cells. To investigate the molecular basis of thymocyte-specific gene expression, we searched for the genomic elements regulating transcription of the mouse pTalpha gene. We now report that expression of the pTalpha gene is primarily controlled by an upstream genomic region, which can drive thymocyte-specific expression of a marker gene in transgenic mice. Within this region, we have identified two specific DNase-hypersensitive sites corresponding to a proximal promoter and an upstream transcriptional enhancer. The pTalpha enhancer appears to function preferentially in pre-T cell lines and binds multiple nuclear factors, including YY1. The enhancer also contains two G-rich stretches homologous to a critical region of the thymocyte-specific lck proximal promoter. Here we show that these sites bind a common nuclear factor and identify it as the zinc finger protein ZBP-89. Our data establish a novel experimental model for thymocyte-specific gene expression and suggest an important role for ZBP-89 in T cell development.