Feng-Qian Li

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. 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.

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) β-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

On the mechanism of DNA transfection : efficient gene transfer without viruses

From an investigation of how transfected DNA navigates from the cell surface to the nucleus, we have developed a transfection method for primary human fibroblasts that approaches the efficiency of viruses. We have visually tracked the subcellular routing of exogenous DNA and find that all cells in an asynchronous population are surprisingly competent in the nuclear uptake of DNA, but two steps practically limit efficient transfection to a minority of cells. First, regardless of the method used to traverse the cell membrane – CaPO4 precipitation, lipofection or electroporation – it appears that nuclear transport of DNA requires routing through endosomes and lysosomes. Apparent abrogation of endosome–lysosome fusion or translocation with microfilament or microtubule toxins, respectively, inhibits the nuclear accumulation of transfected DNA, but interruption of lysosomal function with protease inhibitors promotes it. Second, in normal human fibroblasts, which are refractory to transfection, the exogenous DNA is rapidly excluded from the nucleus, but in HeLa cells, which are readily transfected, there is prolonged nuclear stability of the DNA, indicating the failure in HeLa cells of a mechanism for the elimination of foreign DNA. These observations imply strategies for optimizing gene transfer efficiency in virus-independent approaches to gene therapy.

Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity

β-Catenin functions in both cell–cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of β-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although β-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits β-catenin–mediated transcriptional activation. Here, we identified 14-3-3ε and 14-3-3ζ as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3–binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with β-catenin, causing β-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of β-catenin, thereby antagonizing β-catenin signaling.

Characterization of Mutant Neutrophil Elastase in Severe Congenital Neutropenia

Severe congenital neutropenia is a heritable human disorder characterized by neutropenia and acute myelogenous leukemia. We recently determined that the majority of cases result fromde novo or autosomal dominantly inherited heterozygous mutations in ELA2, encoding neutrophil elastase. Neutrophil elastase is a chymotryptic serine protease localized in granules of neutrophils and monocytes and is the major target of inhibition of the serpin α1-antitrypsin. The mutations causing severe congenital neutropenia consist of amino acid missense substitutions, in-frame deletion, splice donor mutation producing a deletion, splice acceptor mutation causing insertion of novel residues, and protein truncating mutations of the carboxyl terminus resulting from nonsense substitutions and deletions leading to frameshifts. We have expressed 14 mutant forms of neutrophil elastase in vitro and have characterized their biochemical properties. The mutations have variable effects on proteolytic activity, eliminating the possibility that the disease results from haploinsufficiency. There is no evidence that the mutant enzymes are cytotoxic. The mutant enzymes retain vulnerability to inhibition by α1-antitrypsin, but demonstrate variable avidity for interaction with this serpin. Somewhat surprisingly, the mutant enzymes inhibit the wild type enzyme when both are coexpressed within the same cell, suggesting the potential to interfere with normal subcellular trafficking or post-translational processing.

Mediators of activation of fushi tarazu gene transcription by BmFTZ-F1.

Transcriptional activation by many eukaryotic sequence-specific regulators appears to be mediated through transcription factors which do not directly bind to DNA. BmFTZ-F1 is a silkworm counterpart of FTZ-F1, a sequence-specific activator of the fushi tarazu gene in Drosophila melanogaster. We report here the isolation of 18- and 22-kDa polypeptides termed MBF1 and MBF2, respectively, that form a heterodimer and mediate activation of in vitro transcription from the fushi tarazu promoter by BmFTZ-F1. Neither MBF1, MBF2, nor a combination of them binds to DNA. MBF1 interacts with BmFTZ-F1 and stabilizes the BmFTZ-F1-DNA complex. MBF1 also makes direct contact with TATA-binding protein (TBP). Both MBF1 and MBF2 are necessary to form a complex between BmFTZ-F1 and TBP. We propose a model in which MBF1 and MBF2 form a bridge between BmFTZ-F1 and TBP and mediate transactivation by stabilizing the protein-DNA interactions.

Inactivation of Chibby affects function of motile airway cilia

Chibby (Cby) is a conserved component of the Wnt–β-catenin pathway. Cby physically interacts with β-catenin to repress its activation of transcription. To elucidate the function of Cby in vertebrates, we generated Cby−/− mice and found that after 2–3 d of weight loss, the majority of mice die before or around weaning. All Cby−/− mice develop rhinitis and sinusitis. When challenged with Pseudomonas aeruginosa isolates, Cby−/− mice are unable to clear the bacteria from the nasal cavity. Notably, Cby−/− mice exhibit a complete absence of mucociliary transport caused by a marked paucity of motile cilia in the nasal epithelium. Moreover, ultrastructural experiments reveal impaired basal body docking to the apical surface of multiciliated cells. In support of these phenotypes, endogenous Cby protein is localized at the base of cilia. As the phenotypes of Cby−/− mice bear striking similarities to primary ciliary dyskinesia, Cby−/− mice may prove to be a useful model for this condition.

A novel notch protein, N2N, targeted by neutrophil elastase and implicated in hereditary neutropenia.

ABSTRACT Mutations in ELA2, encoding the human serine protease neutrophil elastase, cause cyclic and severe congenital neutropenia, and recent evidence indicates that the mutations alter the membrane trafficking of neutrophil elastase. These disorders feature impaired bone marrow production of neutrophils along with excess monocytes—terminally differentiated lineages corresponding to the two alternative fates of myeloid progenitor cells. We utilized a modified yeast two-hybrid system and identified a new, widely expressed gene, N2N, whose product is homologous to Notch2, that interacts with neutrophil elastase. N2N is a 36-kDa protein distributed throughout the cell and secreted. Its amino-terminal sequence consists of several EGF repeats identical to those of the extracellular region of Notch2, and its carboxyl terminus contains a unique 24-residue domain required for interaction with neutrophil elastase. Neutrophil elastase cleaves N2N within EGF repeats in vitro and in living cells, but the C-terminal domain retards proteolysis. In vitro, N2N represses transcriptional activities of Notch proteins. Disease-causing mutations of neutrophil elastase disrupt the interaction with N2N, impair proteolysis of N2N and Notch2, and interfere with Notch2 signaling, suggesting defective proteolysis of an inhibitory form of Notch as an explanation for the alternate switching of cell fates characteristic of hereditary neutropenia.

Chibby, an Antagonist of the Wnt/β-Catenin Pathway, Facilitates Cardiomyocyte Differentiation of Murine Embryonic Stem Cells

Background— Embryonic stem cell (ESC)–derived cardiomyocytes are anticipated to serve as a useful source for future cell-based cardiovascular disease therapies. Research emphasis is currently focused on determining methods to direct the differentiation of ESCs to a large population of cardiomyocytes with high purity. To this aim, understanding the molecular mechanisms that control ESC-to-cardiomyocyte differentiation should play a critical role in the development of this methodology. The Wnt/β-catenin signaling pathway has been implicated in both embryonic cardiac development and in vitro ESC differentiation into cardiomyocytes. Chibby is a recently identified nuclear protein that directly binds to β-catenin and antagonizes its transcriptional activity. Methods and Results— Chibby was ubiquitously expressed in early stages of ESC differentiation but upregulated during cardiomyocyte specification. Of interest, the Chibby gene promoter has multiple binding sites for the cardiac-specific homeodomain protein Nkx2.5, and its promoter activity was indeed positively regulated by Nkx2.5. Furthermore, overexpression of Chibby increased cardiac differentiation of ESCs, whereas loss of Chibby by RNAi impaired cardiomyocyte differentiation. Conclusions— These data illustrate the regulation and function of Chibby in facilitating cardiomyocyte differentiation from ESCs. By revealing molecular mechanisms that control ESC-to-cardiomyocyte differentiation, this study will allow for the future development of technologies to improve cardiomyocyte differentiation from ESCs.

An Oncogenic Hub: β-Catenin as a Molecular Target for Cancer Therapeutics

The Wnt/beta-catenin signaling pathway plays diverse roles in embryonic development and in maintenance of organs and tissues in adults. Activation of this signaling cascade inhibits degradation of the pivotal component beta-catenin, which in turn stimulates transcription of downstream target genes. Over the past two decades, intensive worldwide investigations have yielded considerable progress toward understanding the cellular and molecular mechanisms of Wnt signaling and its involvement in the pathogenesis of a range of human diseases. Remarkably, beta-catenin signaling is aberrantly activated in greater than 70% of colorectal cancers and to a lesser extent in other tumor types, promoting cancer cell proliferation, survival and migration. Accordingly, beta-catenin has gained recognition as an enticing molecular target for cancer therapeutics. Disruption of protein-protein interactions essential for beta-catenin activity holds immense promise for the development of novel anti-cancer drugs. In this review, we focus on the regulation of beta-catenin-dependent transcriptional activation and discuss potential therapeutic opportunities to block this signaling pathway in cancer.