Genome-wide map of nuclear protein degradation shows NCoR1 turnover as a key to mitochondrial gene regulation.

Transcription factor activity and turnover are functionally linked, but the global patterns by which DNA-bound regulators are eliminated remain poorly understood. We established an assay to define the chromosomal location of DNA-associated proteins that are slated for degradation by the ubiquitin-proteasome system. The genome-wide map described here ties proteolysis in mammalian cells to active enhancers and to promoters of specific gene families. Nuclear-encoded mitochondrial genes in particular correlate with protein elimination, which positively affects their transcription. We show that the nuclear receptor corepressor NCoR1 is a key target of proteolysis and physically interacts with the transcription factor CREB. Proteasome inhibition stabilizes NCoR1 in a site-specific manner and restrains mitochondrial activity by repressing CREB-sensitive genes. In conclusion, this functional map of nuclear proteolysis links chromatin architecture with local protein stability and identifies proteolytic derepression as highly dynamic in regulating the transcription of genes involved in energy metabolism.

Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.

Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue.

Protein kinase CK1alphaLS promotes vascular cell proliferation and intimal hyperplasia.

Protein kinase CK1alpha regulates several fundamental cellular processes including proliferation and differentiation. Up to four forms of this kinase are expressed in vertebrates resulting from alternative splicing of exons; these exons encode either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein. Whereas the L-insert is known to target the kinase to the nucleus, the functional significance of nuclear CK1alphaLS has been unclear. Here we demonstrate that selective L-insert-targeted short hairpin small interfering RNA-mediated knockdown of CK1alphaLS in human vascular endothelial cells and vascular smooth muscle cells impairs proliferation and abolishes hydrogen peroxide-stimulated proliferation of vascular smooth muscle cells, with the cells accumulating in G(0)/G(1). In addition, selective knockdown of CK1alphaLS in cultured human arteries inhibits vascular activation, preventing smooth muscle cell proliferation, intimal hyperplasia, and proteoglycan deposition. Knockdown of CK1alphaLS results in the harmonious down-regulation of its target substrate heterogeneous nuclear ribonucleoprotein C and results in the altered expression or alternative splicing of key genes involved in cellular activation including CXCR4, MMP3, CSF2, and SMURF1. Our results indicate that the nuclear form of CK1alpha in humans, CK1alphaLS, plays a critical role in vascular cell proliferation, cellular activation, and hydrogen peroxide-mediated mitogenic signal transduction.

Dysfunctional expansion of hematopoietic stem cells and block of myeloid differentiation in lethal sepsis.

Severe sepsis is one of the leading causes of death worldwide. High mortality rates in sepsis are frequently associated with neutropenia. Despite the central role of neutrophils in innate immunity, the mechanisms causing neutropenia during sepsis remain elusive. Here, we show that neutropenia is caused in part by apoptosis and is sustained by a block of hematopoietic stem cell (HSC) differentiation. Using a sepsis murine model, we found that the human opportunistic bacterial pathogen Pseudomonas aeruginosa caused neutrophil depletion and expansion of the HSC pool in the bone marrow. "Septic" HSCs were significantly impaired in competitive repopulation assays and defective in generating common myeloid progenitors and granulocyte-monocyte progenitors, resulting in lower rates of myeloid differentiation in vitro and in vivo. Delayed myeloid-neutrophil differentiation was further mapped using a lysozyme-green fluorescent protein (GFP) reporter mouse. Pseudomonas's lipopolysaccharide was necessary and sufficient to induce myelosuppresion and required intact TLR4 signaling. Our results establish a previously unrecognized link between HSC regulation and host response in severe sepsis and demonstrate a novel role for TLR4.

Mixed chimerism, lymphocyte recovery, and evidence for early donor-specific unresponsiveness in patients receiving combined kidney and bone marrow transplantation to induce tolerance.

BACKGROUND: We have previously reported operational tolerance in patients receiving human leukocyte antigen-mismatched combined kidney and bone marrow transplantation (CKBMT). We now report on transient multilineage hematopoietic chimerism and lymphocyte recovery in five patients receiving a modified CKBMT protocol and evidence for early donor-specific unresponsiveness in one of these patients. METHODS: Five patients with end-stage renal disease received CKBMT from human leukocyte antigen-mismatched, haploidentical living-related donors after modified nonmyeloablative conditioning. Polychromatic flow cytometry was used to assess multilineage chimerism and lymphocyte recovery posttransplant. Limiting dilution analysis was used to assess helper T-lymphocyte reactivity to donor antigens. RESULTS: Transient multilineage mixed chimerism was observed in all patients, but chimerism became undetectable by 2 weeks post-CKBMT. A marked decrease in T- and B-lymphocyte counts immediately after transplant was followed by gradual recovery. Initially, recovering T cells were depleted of CD45RA+/CD45RO(-) "naïve-like" cells, which have shown strong recovery in two patients, and CD4:CD8 ratios increased immediately after transplant but then declined markedly. Natural killer cells were enriched in the peripheral blood of all patients after transplant.For subject 2, a pretransplant limiting dilution assay revealed T helper cells recognizing both donor and third-party peripheral blood mononuclear cells. However, the antidonor response was undetectable by day 24, whereas third-party reactivity persisted. CONCLUSION: These results characterize the transient multilineage mixed hematopoietic chimerism and recovery of lymphocyte subsets in patients receiving a modified CKBMT protocol. The observations are relevant to the mechanisms of donor-specific tolerance in this patient group.

MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors.

Lineage specification is a critical issue in developmental and regenerative biology. We hypothesized that microRNAs (miRNAs) are important participants in those processes and used the poorly understood regulation of megakaryocyte-erythrocyte progenitors (MEPs) in hematopoiesis as a model system. We report here that miR-150 modulates lineage fate in MEPs. Using a novel methodology capable of profiling miRNA expression in small numbers of primary cells, we identify miR-150 as preferentially expressed in the megakaryocytic lineage. Through gain- and loss-of-function experiments, we demonstrate that miR-150 drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo. Moreover, we identify the transcription factor MYB as a critical target of miR-150 in this regulation. These experiments show that miR-150 regulates MEP fate, and thus establish a role for miRNAs in lineage specification of mammalian multipotent cells.

Bone marrow transplantation generates immature oocytes and rescues long-term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure.

PURPOSE: Although early menopause frequently occurs in female cancer patients after chemotherapy (CTx), bone marrow (BM) transplantation (BMT) has been linked to an unexplained return of ovarian function and fertility in some survivors. Studies modeling this in mice have shown that BMT generates donor-derived oocytes in CTx-treated recipients. However, a subsequent report claimed that ovulated eggs are not derived from BM and that BM-derived oocytes reported previously are misidentified immune cells. This study was conducted to further clarify the impact of BMT on female reproductive function after CTx using a preclinical mouse model. METHODS: Female mice were administered CTx followed by BMT using coat color-mismatched female donors. After housing with males, the number of pregnancies and offspring genotype were recorded. For cell tracking, BM from germline-specific green fluorescent protein-transgenic mice was transplanted into CTx-treated wild-type recipients. Immune cells were sorted from blood and analyzed for germline markers. RESULTS: BMT rescued long-term fertility in CTx-treated females, but all offspring were derived from the recipient germline. Cell tracking showed that donor-derived oocytes were generated in ovaries of recipients after BMT, and two lines of evidence dispelled the claim that these oocytes are misidentified immune cells. CONCLUSION: These data from a preclinical mouse model validate a testable clinical strategy for preserving or resurrecting ovarian function and fertility in female cancer patients after CTx, thus aligning with recommendations of the 2005 National Cancer Institute Breast Cancer Progress Review Group and President's Cancer Panel to prioritize research efforts aimed at improving the quality of life in cancer survivors.

Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis.

The zinc finger transcription factor GATA-2 plays a fundamental role in generating hematopoietic stem-cells in mammalian development. Less well defined is whether GATA-2 participates in adult stem-cell regulation, an issue we addressed using GATA-2 heterozygote mice that express reduced levels of GATA-2 in hematopoietic cells. While GATA-2+/- mice demonstrated decreases in some colony-forming progenitors, the most prominent changes were observed within the stem-cell compartment. Heterozygote bone marrow had a lower abundance of Lin(-)c-kit(+)Sca-1(+)CD34- cells and performed poorly in competitive transplantation and quantitative week-5 cobblestone area-forming cell (CAFC) assays. Furthermore, a stem-cell-enriched population from GATA1+/- marrow was more quiescent and exhibited a greater frequency of apoptotic cells associated with decreased expression of the anti-apoptotic gene Bcl-xL. Yet the self-renewal potential of the +/- stem-cell compartment, as judged by serial transplantations, was unchanged. These data indicate compromised primitive cell proliferation and survival in the setting of a lower GATA-2 gene dose without a change in the differentiation or self-renewal capacity of the stem-cells that remain. Thus, GATA-2 dose regulates adult stem-cell homeostasis by affecting select aspects of stem cell function.

Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells.

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion.

Differential expression of cell surface antigens on subsets of CD4+ and CD8+ T cells.

BACKGROUND: The assessment of relative antigen density on T cell subsets is a feature of antigen expression that is infrequently characterized. Defining phenotypic differences is a first step in understanding associated differences in function. Additionally, a better understanding of T cell heterogeneity may aid in clinical diagnoses. MATERIAL/METHODS: To further elucidate phenotypic differences of T cell subsets, and begin to determine what information relative antigen density contributes to immunology, we analyzed normal human peripheral blood T cells for a variety of immunophenotypic (CD2, CD3, CD4, CD5, CD7, CD8, CD45RA, CD45RO, TCR alpha beta) and light scatter characteristics using 6 color flow cytometry. T-cell leukemia specimens were also analyzed. RESULTS: Our data show that statistically significant immunophenotypic differences exist between subsets of human CD4 and CD8 T cells. Normal T cells express different levels of relative antigen density for some antigens compared to malignant T cells. CONCLUSIONS: Significant differences are seen in relative antigen density for several cell surface markers between CD4+ and CD8+ T cells. Neither donor source nor flow cytometric calibration account for these differences. The data are applied to specimens from patients with T-lineage acute lymphoblastic leukemia to show how antigen density can be used clinically in aiding to diagnose disease. The data presented here can be used to further investigate these cell populations for functional differences.