Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells.

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Hematopoietic stem cell enhancer: a powerful tool in stem cell biology.

There has been considerable interest in identifying a cis-regulatory element that targets gene expression to stem cells. Such an element, termed stem cell enhancer, holds the promise of providing important insights into the transcriptional programs responsible for inherent stem cell-specific properties such as self-renewal capacity. The element also serves as a molecular handle for stem cell-specific marking, transgenesis and gene targeting, thereby becoming invaluable to stem cell research. A series of candidate enhancers have been identified for hematopoietic stem cells (HSCs). This review summarizes currently known HSC enhancers with emphasis on an intronic enhancer in the Runx1 gene which is essential for the generation and maintenance of HSCs. The element, named eR1 (+24m), is active specifically in HSCs, but not in progenitors, and is hence the most definitive HSC enhancer.

Three-dimensional imaging of whole midgestation murine embryos shows an intravascular localization for all hematopoietic clusters.

Hematopoietic cell clusters associated with the midgestation mouse aorta, umbilical and vitelline arteries play a pivotal role in the formation of the adult blood system. Both genetic and live-imaging data indicate that definitive hematopoietic progenitor/stem cells (visualized as clusters) are generated from hemogenic endothelium. A 3-dimensional (3-D) whole embryo immunostaining and imaging technique has allowed quantitation and cartographic mapping of intravascular hematopoietic clusters. During this period the vitelline artery is most extensively remodeled, and several reports have suggested that vitelline remodeling leads to extravascular hematopoietic cluster emergence. Whether the earliest definitive progenitors/stem cells are intra or extra vascular could influence the process by which these cells migrate to the next hematopoietic territory, the fetal liver. Hence, by 3-D imaging we more closely examined hematopoietic clusters in the vitelline and associated connected small vessels and show here that hematopoietic clusters (particularly large clusters) are intravascular during the period of vascular remodeling.

A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells.

Runx1 is essential for the generation of hematopoietic stem cells (HSCs) and is frequently mutated in human leukemias. However, the cis-regulatory mechanisms modulating the Runx1 gene expression remain to be elucidated. Herewith, we report the identification of an intronic Runx1 enhancer, Runx1 +24 mouse conserved noncoding element (mCNE), using a combinatorial in silico approach involving comparative genomics and retroviral integration sites mapping. The Runx1 +24 mCNE was found to possess hematopoietic-specific enhancer activity in both zebrafish and mouse models. Significantly, this enhancer is active specifically in hemogenic endothelial cells (ECs) at sites where the de novo generation of HSCs occurs. The activity of this enhancer is also strictly restricted to HSCs within the hematopoietic compartment of the adult bone marrow. We anticipate that Runx1 +24 mCNE HSC enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic ECs/HSCs behavior in vivo, and consequently become an invaluable tool for research on stem cell and cancer biology.

Retroviral integration sites (RIS) mark cis-regulatory elements.

Transcription in multicellular eukaryotic organisms involves an elaborate orchestration of the core promoter and cis-regulatory elements to drive spatiotemporally and quantitatively correct gene expression. Unlike promoters found immediately upstream of protein-coding genes, the positions of distally located cis-regulatory elements relative to a gene of interest are difficult to define. As such, the identification and characterization of these regulatory elements has proved to be challenging. To this end, we propose a combinatorial in silico approach involving retroviral integration sites (RIS) mapping together with predicted matrix attachment regions (MARs) mapping and an already well-established comparative genomics approach, to enhance the prediction of potential cis-regulatory elements. Predicted elements can be validated by further investigations to ascertain their functions. In view of the abundance of electronically available RIS information, RIS mapping has an unrealized potential to aid in the discovery of novel cis-regulatory elements.

cDNA cloning of Runx family genes from the pufferfish (Fugu rubripes).

The Runx family genes are involved in hematopoiesis, osteogenesis and neuropoiesis, and mutations in these genes have been frequently associated with human hereditary diseases and cancers. Here we report the cDNA cloning of the full Runx gene family of the pufferfish (Fugu rubripes), which comprises frRunx1, frRunx2, frRunx3, frRunt and frCbfb. Fugu is evolutionarily distant from mammals, thus the annotation of the frRunx family genes greatly facilitates comparative genomics approaches. Protein sequence comparison revealed that the fugu genes show high conservation in the Runt domain and PY and VWRPY motifs. frRunx1 had an extra stretch of eight histidine residues, while frRunx2 lacked the poly-glutamine/-alanine stretch that is a hallmark of the mammalian Runx2 genes. Analysis of the promoter regions revealed high conservation of the binding sites for transcription factors, including Runx sites in the P1 promoters. Abundant CpG dinucleotides in the P2 promoter regions were also detected. The expression patterns of the frRunx family genes in various tissues showed high similarity to those of the mammalian Runx genes. The genomic structures of the fugu and mammalian Runx genes are largely conserved except for a split exon 2 in frRunx1 and an extra exon in the C-terminal region of frRunx3 that is missing in mammalian Runx3 genes. The similarities and differences between the Runx family genes of fugu and mammals will improve our understanding of the functions of these proteins.

Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration.

The recently cloned gene Nogo, whose alternative splice products correspond to the antigenic target of the central nervous system (CNS) regeneration enhancing monoclonal antibody IN-1, codes for membrane proteins enriched in brain, particularly in oligodendrocytes. The 66-amino acid extracellular domain of Nogo (Nogo-66) interacts with a high-affinity receptor (NgR), a glycosylphosphatidylinositol (GPI)-linked protein with multiple leucine-rich repeats. The amino terminal cytoplasmic domain of Nogo appears to have a general cellular growth inhibitory effect. Nogo-66, on the other hand, specifically retards neurite outgrowth and induces growth cone collapse, possibly through its interaction with NgR and as yet unidentified transmembrane coreceptors. Recent results also suggest that Nogo expression may induce apoptosis in tumor cells. Together, these proteins provide new molecular handles for the design of therapeutic interventions for CNS injuries and neurodegenerative diseases, as well as possible leads to anticancer strategies.