Unique N-terminal sequences in two Runx1 isoforms are dispensable for Runx1 function

Abstract Background The Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein. Results To address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform. Conclusions Our results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production

Induced developmental arrest of early hematopoietic progenitors leads to the generation of leukocyte stem cells

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status. We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy

Additional file 1: of Unique N-terminal sequences in two Runx1 isoforms are dispensable for Runx1 function

Strategy used to generate Runx1DP1:P2TAA mutant allele by sequential gene targeting. Schematic representation of the targeting strategy used to generate the Runx1DP1:P2TAA allele. Open and closed boxes represent the 5′ untranslated region (UTR) and coding region in exon I and II, respectively. The neor and tk indicate neomycin resistance and thymidine kinase genes, respectively. Triangles represent loxP sequences. To select ES cells with G418 after transfection of the target vector for the Runx1 P2TAA mutation, the neor gene was removed from ES clones harboring the Runx1 +/P1N genotype, thus generating ES clones harboring the Runx1 +/ΔP1 genotype. Cells were transfected with the target vector for the Runx1 P2TAA mutation and clones that underwent homologous recombination were isolated. To screen for whether the Runx1 or Runx1DP1 allele was targeted to the Runx1 P2TAA mutation, ES clones were transduced with a retroviral vector encoding Cre recombinase and screened by PCR for an inverted recombination event between loxP sequences in opposite directions. ES clones harboring the Runx1 +/ΔP1: P2TAAN genotype were isolated. Primers are indicated as red arrowheads. Gel image on the right shows detection of inverted recombination in clones 3–8 and 9–8. The neo r gene was removed by transient transfection of Cre recombinase to isolate ES clones harboring the Runx1 +/ΔP1:P2TAA genotype. (PDF 209 kb

Additional file 2: of Unique N-terminal sequences in two Runx1 isoforms are dispensable for Runx1 function

Detection of a cryptic TSS that mapped to a region 220 bp downstream of the canonical TSS in the public FANTOM5 database. Image of FANTOM5 web browser showing canonical and cryptic transcriptional start site (TSS), which are marked with arrow heads, for P2-Runx1 transcript. Red line indicates a genomic region that was deleted in the Runx1P2TAG allele. Numbers represent nucleotide positons according to mm9 reference. (PDF 84 kb

A Novel Gene Essential for the Development of Single Positive Thymocytes▿

A critical step during intrathymic T-cell development is the transition of CD4+ CD8+ double-positive (DP) cells to the major histocompatibility complex class I (MHC-I)-restricted CD4− CD8+ and MHC-II-restricted CD4+ CD8− single-positive (SP) cell stage. Here, we identify a novel gene that is essential for this process. Through the T-cell phenotype-based screening of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we established a mouse line in which numbers of CD4 and CD8 SP thymocytes as well as peripheral CD4 and CD8 T cells were dramatically reduced. Using linkage analysis and DNA sequencing, we identified a missense point mutation in a gene, E430004N04Rik (also known as themis), that does not belong to any known gene family. This orphan gene is expressed specifically in DP and SP thymocytes and peripheral T cells, whereas in mutant thymocytes the levels of protein encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice, and their phenotype was virtually identical to that of the ENU mutant mice, thereby confirming that this gene is essential for the development of SP thymocytes

Priming of lineage-specifying genes by Bcl11b is required for lineage choice in post-selection thymocytes

CD4 and CD8 T cells develop in the thymus with their transcription programs controlled by ThPOK and Runx3, respectively. Here the authors show that a pre-commitment event modulated by the transcription factor, Bcl11b, is required for the proper expression of ThPOK and Runx3 and correct CD4/CD8 lineage commitment

Maturation of mTECs was not affected in Foxn1-Stat3-CKO.

<p>Cryostat sections of the thymus were stained with antibodies directed to K14 (green) and UEA1 (red) (A), and with antibodies to ERTR5 (red) and AIRE (green) (B). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p