Subversion of T lineage commitment by PU.1 in a clonal cell line system

Abstract

Specification of mammalian T lymphocytes involves prolonged developmental plasticity even after lineage-specific gene expression begins. Expression of transcription factor PU.1 may maintain some myeloid-like developmental alternatives until commitment. Commitment could reflect PU.1 shutoff, resistance to PU.1 effects, and/or imposition of a suicide penalty for diversion. Here, we describe subclones from the SCID.adh murine thymic lymphoma, adh.2C2 and adh.6D4, that represent a new tool for probing these mechanisms. PU.1 can induce many adh.2C2 cells to undergo diversion to a myeloid-like phenotype, in an all-or-none fashion with multiple, coordinate gene expression changes; adh.6D4 cells resist diversion, and most die. Diversion depends on the PU.1 Ets domain but not on known interactions in the PEST or Q-rich domains, although the Q-rich domain enhances diversion frequency. Protein kinase C/MAP kinase stimulation can make adh.6D4 cells permissive for diversion without protecting from suicide. These results show distinct roles for regulated cell death and another stimulation-sensitive function that establishes a threshold for diversion competence. PU.1 also diverts normal T-cell precursors from wild type or Bcl2-transgenic mice to a myeloid-like phenotype, upon transduction in short-term culture. The adh.2C2 and adh.6D4 clones thus provide an accessible system for defining mechanisms controlling developmental plasticity in early T-cell development.

Downregulation of the transcription factor KLF4 is required for the lineage commitment of T cells

“…The E protein (also named helix-loop-helix protein) and Notch signaling cooperate to promote early T cell development [33]. HEBAlt, a long form of the E protein HEB gene, is specifically expressed in lymphoid precursor cells [34]. In thymic precursors, HEBAlt collaborates with Notch signals to promote early T cell development by suppressing B cell or myeloid potential [35][36].…”

“…Relative expression levels were normalized to Gapdh or β-actin transcript levels and calculated using the 2 -∆∆CT method. The sequences of the primers used have been previously published [1,20,34,[47][48][49][50] and are listed under Supplementary information, Table S1.…”

The Basic Helix-Loop-Helix Transcription Factor HEBAlt Is Expressed in Pro-T Cells and Enhances the Generation of T Cell Precursors

“…In this report we describe a new form of HEB called HEBAlt, which is expressed specifically in early T cell precursors. The distinctive expression pattern of this factor in thymocytes has already prompted us to use it as a reference sample in a few studies (22)(23)(24). In this study, however, we present the first detailed description of HEBAlt, and show that HEBAlt is specifically required for efficient generation of T cell precursors.…”

Transcriptional control of granulocyte and monocyte development

“…In addition to favoring monopoiesis over granulopoiesis, increased PU.1 expression also favors myeloid over lymphoid development: expression of exogenous PU.1 in PU.1(À/À) progenitors induces B cells at lower levels and monocytes at higher levels (DeKoter and Singh, 2000); exogenous PU.1 converts B or T cells to monocyte/macrophages but not granulocytes (Xie and Graf, 2004;Dionne et al, 2005;Laiosa et al, 2006); and PU.1 knockdown in embryonic stem cell-derived CD34 þ cells favors B-cell formation (Zou et al, 2006). The ability of both C/EBPs and PU.1 to reprogram lymphoid cells to the monocyte lineage likely reflects the ability of C/EBPs to bind to and activate the PU.1 promoter and distal enhancer.…”