The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

No novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide CODEHOP-based primers.

Novel viruses might be responsible for numerous disease cases with unknown etiology. In this study, we screened 1800 nasopharyngeal samples from adult outpatients with respiratory disease symptoms and healthy individuals. We employed a reverse transcription (RT)-PCR assay and CODEHOP-based primers (CT12-mCODEHOP) previously developed to recognize known and unknown corona- and toroviruses. The CT12-mCODEHOP assay detected 42.0 % (29/69) of samples positive for human coronaviruses (HCoV), including HCoV-229 (1/16), HCoV-NL63 (9/17), and HCoV-OC43 (19/36), and additionally HCoV-HKU1 (3), which was not targeted by the diagnostic real-time PCR assays. No other coronaviruses were identified in the analyzed samples.

Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

Regulation of hematopoietic stem cell fate by the ubiquitin proteasome system.

Hematopoietic stem cells (HSCs) residing in the bone marrow generate mature blood cells throughout the life of the organism. This is accomplished by careful regulation of HSC activity to balance quiescence, self-renewal and differentiation. Studies of the molecular mechanisms governing HSC maintenance have mostly focused on the role of signaling and transcriptional processes. However, it has recently been demonstrated that protein regulation via the ubiquitin proteasome system (UPS) is crucial for normal HSC function; the loss of which can lead to transformation and leukemogenesis. The effective use of a general and reversible inhibitor of the UPS, bortezomib, in treating mantle cell lymphoma and multiple myeloma has demonstrated that targeting the UPS has therapeutic potential. Thus, understanding the emerging field of how the UPS regulates HSC activity may lead to novel targets for therapy of leukemia.

Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses.

The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.

Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.

Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2(+/-) mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.

Regulation of hematopoietic stem cell differentiation by a single ubiquitin ligase-substrate complex.

Hematopoietic stem cell (HSC) differentiation is regulated by cell-intrinsic and cell-extrinsic cues. In addition to transcriptional regulation, post-translational regulation may also control HSC differentiation. To test this hypothesis, we visualized the ubiquitin-regulated protein stability of a single transcription factor, c-Myc. The stability of c-Myc protein was indicative of HSC quiescence, and c-Myc protein abundance was controlled by the ubiquitin ligase Fbw7. Fine changes in the stability of c-Myc protein regulated the HSC gene-expression signature. Using whole-genome genomic approaches, we identified specific regulators of HSC function directly controlled by c-Myc binding; however, adult HSCs and embryonic stem cells sensed and interpreted c-Myc-regulated gene expression in distinct ways. Our studies show that a ubiquitin ligase-substrate pair can orchestrate the molecular program of HSC differentiation.

Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid.

On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pre-T cell receptor (pre-TCR), which orchestrates differentiation, cell proliferation, and survival. The exact mechanism of pre-TCR-mediated suppression of T cell death remains unclear and controversial. Here, we identify Bim and Bid, 2 members of the BH3-only group of the BCL2 family, as important regulators of pre-T cell death. Both factors are highly expressed in proapoptotic thymocytes and their expression is suppressed on signaling through the pre-TCR. Their expression is directly regulated by the transcription factors FoxO3a and p53. Bid expression and p53 activity are related to the ongoing rearrangement of the TCR loci and induced DNA damage responses. Bim expression and FoxO3a nuclear translocation are directly controlled by the pre-TCR by means of its downstream kinase Akt/PKB. Interestingly, deletion of either gene on a pre-TCR(-/-) background rescues survival, but fails to induce further progenitor differentiation uncoupling the 2 processes.