No abstract

Multicellular systems develop from single cells through distinct lineages. However, current lineage tracing approaches scale poorly to whole, complex organisms. Here we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable, and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease.

The function of metabolic state in stemness is poorly understood. Mouse embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent states representing the inner cell mass (ICM) and epiblast embryos. Human embryonic stem cells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic difference between these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalent in their energy production, dynamically switching from glycolysis to mitochondrial respiration on demand. Despite having a more developed and expanding mitochondrial content, EpiSC/hESC have low mitochondrial respiratory capacity due to low cytochrome c oxidase (COX) expression. Similarly, in vivo epiblasts suppress COX levels. These data reveal EpiSC/ hESC functional similarity to the glycolytic phenotype in cancer (Warburg effect). We further show that hypoxiainducible factor 1a (HIF1a) is sufficient to drive ESC to a glycolytic Activin/Nodal-dependent EpiSC-like stage. This metabolic switch during early stem-cell development may be deterministic.

We report the discovery of the GATA2 gene as a new myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) predisposition gene. We found the same, novel heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS/AML in three families, and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS/AML family. The mutations reside within the second zinc finger of GATA2 which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutants on transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counselling, selection of related bone marrow transplant donors, and development of therapies.

Low oxygen levels have shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia inducible factor (HIF), can induce a hESC-like transcriptional program, including the iPSC inducers, OCT4, NANOG, SOX2, KLF4, cMYC and miRNA-302 in eleven cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver and breast tumors). Further, non-degradable forms of HIFα, combined with the traditional iPSC inducers are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4- and HIF1α-positive regions. Further, NANOG and OCT4 expression positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells neurospheres and hESC markers were induced in hypoxia but not in normoxia. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathological conditions.

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/ AML) is an autosomal dominant familial platelet disorder characterized by thrombocytopenia and a propensity to develop AML. Mutation analyses of RUNX1 in 3 families with FPD/AML showing linkage to chromosome 21q22.1 revealed 3 novel heterozygous point mutations (K83E, R135fsX177 (IVS4 ؉ 3delA), and Y260X). Functional investigations of the 7 FPD/ AML RUNX1 Runt domain point mutations described to date (2 frameshift, 2 nonsense, and 3 missense mutations) were performed. Consistent with the position of the mutations in the Runt domain at the RUNX1-DNA interface, DNA binding of all mutant RUNX1 proteins was absent or significantly decreased. In general, missense and nonsense RUNX1 proteins retained the ability to heterodimerize with PEBP2␤/CBF␤ and inhibited transactivation of a reporter gene by wild-type RUNX1. Colocalization of mutant RUNX1 and PEBP2␤/CBF␤ in the cytoplasm was observed. These results suggest that the sequestration of PEBP2␤/CBF␤ by mutant RUNX1 may cause the inhibitory effects. While haploinsufficiency of RUNX1 causes FPD/AML in some families (deletions and frameshifts), mutant RUNX1 proteins (missense and nonsense) may also inhibit wild-type RUNX1, possibly creating a higher propensity to develop leukemia. This is consistent with the hypothesis that a second mutation has to occur, either in RUNX1 or another gene, to cause leukemia among individuals harboring RUNX1 FPD/AML mutations and that the propensity to acquire these additional mutations is determined, at least partially, by the initial RUNX1 mutation. IntroductionThe most frequent mutations associated with leukemia are recurrent somatic chromosomal translocations or inversions, many of which involve the polyomavirus enhancer-binding protein or core-binding factor transcriptional regulation complex (PEBP2/CBF). Several translocations involve the ␣ subunit of this complex, the RUNX1 gene (also called AML1, CBF␣2, or PEBP2␣B) on chromosome 21q22.1 (t(8;21), t(3;21), and t(12;21)). Additionally, the ␤ subunit of the complex, PEBP2␤ also called CBF␤, is disrupted in inv(16)(p13;q22). 1 An abundance of evidence points to the existence of genes that predispose to hematologic malignancies. However, large multiple-generation families with hematologic malignancies alone are rare. 2 Only 2 loci for familial hematologic malignancies have been identified to date, 1 on chromosome 21q22.1 3 and the other on 16q22. 4,5 These loci contain RUNX1 and PEBP2␤/CBF␤, respectively.Studies of families that demonstrate single-gene inheritance for leukemia predisposition should help to identify the genes and mechanisms involved in the first steps of leukemia development. The autosomal dominant familial platelet disorder (FPD)/ AML (acute myelogenous leukemia; Online Mendelian Inheritance in Man no. 601399) is a good model to validate this hypothesis because, in addition to developing thrombocytopenia, patients show a propensity for progression to myelodysplasia and acute myeloid leuke...

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic 1,2 . We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.Low neutrophil numbers lead to opportunistic infections. There are two hereditary human neutropenia syndromes: cyclic hematopoiesis 4 , comprising three-week oscillations of blood cells, and SCN 3 , consisting of statically low neutrophil counts progressing to leukemia. Heterozygous mutations of ELA2 cause cyclic hematopoiesis and about two-thirds of SCN cases. Mutations in WAS (different from those that cause Wiskott-Aldrich thrombocytopenia) also cause SCN 5 . Owing to its severity, SCN usually arises from new mutations, and additional genes associated with neutropenia have not yet been identified.

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.