TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation.

As key regulators in cellular functions, microRNAs (miRNAs) themselves need to be tightly controlled. Lin28, a pluripotency factor, was reported to downregulate let-7 miRNA by inducing uridylation of let-7 precursor (pre-let-7). But the enzyme responsible for the uridylation remained unknown. Here we identify a noncanonical poly (A) polymerase, TUTase4 (TUT4), as the uridylyl transferase for pre-let-7. Lin28 recruits TUT4 to pre-let-7 by recognizing a tetra-nucleotide sequence motif (GGAG) in the terminal loop. TUT4 in turn adds an oligouridine tail to the pre-let-7, which blocks Dicer processing. Other miRNAs with the same sequence motif (miR-107, -143, and -200c) are regulated through the same mechanism. Knockdown of TUT4 and Lin28 reduces the level of stem cell markers, suggesting that they are required for stem cell maintenance. This study uncovers the role of TUT4 and Lin28 as specific suppressors of miRNA biogenesis, which has implications for stem cell research and cancer biology.

Notch signaling promotes the generation of EphrinB1-positive intestinal epithelial cells.

BACKGROUND & AIMS: The intestinal epithelium consists of EphB2-positive proliferative basal cryptic cells and EphrinB1-positive, postmitotic differentiated cells. We investigated the effects of Notch signaling on formation of the EphB2-EphrinB1 boundary using mouse and tissue culture models. METHODS: We created mice in which Mind bomb-1 (Mib1), an essential E3 ubiquitin ligase that activates Notch ligands, was inactivated specifically in the intestinal epithelia (Vil-Cre;Mib1(f/f)); Notch is, therefore, inactivated in this tissue. We also studied the effects of different inhibitors on intestinal epithelial cells (IEC-6) that express activated Notch. Tissues and cells were analyzed by immunohistochemical and immunoblot analyses. RESULTS: The intestinal epithelia of Vil-Cre;Mib1(f/f) mice had reduced numbers of EphrinB1-positive cells, compared with controls, but increases in EphB2-positive cells; beta-catenin was activated in these cells. These phenotypes were reversed by expression of a constitutively active form of Notch1. In the IEC-6 cells, Notch signaling activated the expression of EphrinB1 in an Hes1-independent manner, but down-regulated the expression of EphB2 through the GSK3beta-mediated inhibition of beta-catenin. CONCLUSIONS: Notch signaling regulates formation of the EphB2-EphrinB1 boundary in the mouse intestinal epithelium.

Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development.

Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1-null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

Defective Notch activation in microenvironment leads to myeloproliferative disease.

Despite the great importance of nonhematopoietic cells constituting the microenvironment for normal hematopoiesis, the cellular interactions between nonhematopoietic cells themselves are largely unknown. Using the Cre-loxP system in mice to inactivate Mind bomb-1 (Mib1), an essential component for Notch ligand endocytosis, here we show that the development of an MPD is dependent on defective Notch activation in the microenvironment. Our 2 independent Mib1 conditional knockout (CKO) mouse lines each developed a myeloproliferative disease (MPD), with gradual accumulations of immature granulocytes. The mutant mice showed hepatosplenomegaly, anemia, granulocytosis, and leukocyte infiltration in multiple organs and finally died at approximately 20 weeks of age. We were surprised to find that the transplantation of wild-type bone marrow cells into the Mib1-null microenvironment resulted in a de novo MPD. Moreover, by introducing the constitutively active intracellular domain of Notch1 in the Mib1-null background, we show that active Notch1 expression in the Mib1-null microenvironment significantly suppressed the disease progression, suggesting that the MPD development in the Mib1 CKO mice is due to defective Notch activation in the nonhematopoietic cells. These findings demonstrate that normal hematopoiesis absolutely requires Notch activation through the Notch ligand-receptor interaction between microenvironmental cells themselves and shed light on the microenvironment that fosters hematopoietic disorders.

Mind bomb-1 is essential for intraembryonic hematopoiesis in the aortic endothelium and the subaortic patches.

Intraembryonic hematopoiesis occurs at two different sites, the floor of the aorta and subaortic patches (SAPs) of the para-aortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region. Notch1 and RBP-jkappa are critical for the specification of hematopoietic stem cells (HSCs) in Notch signal-receiving cells. However, the mechanism by which Notch signaling is triggered from the Notch signal-sending cells to support embryonic hematopoiesis remains to be determined. We previously reported that Mind bomb-1 (Mib1) regulates Notch ligands in the Notch signal-sending cells (B. K. Koo, M. J. Yoon, K. J. Yoon, S. K. Im, Y. Y. Kim, C. H. Kim, P. G. Suh, Y. N. Jan, and Y. Y. Kong, PLoS ONE 2:e1221, 2007). Here, we show that intraembryonic hematopoietic progenitors were absent in the P-Sp of Mib1(-/-) embryos, whereas they were partly preserved in the Tie2-cre; Mib1(f)(/f) P-Sps, suggesting that Mib1 plays a role in the endothelium and the SAPs. Interestingly, dll1 and dll4/Jag1 are expressed in the SAPs and the endothelium of the AGM, respectively, where mib1 is detected. Indeed, Notch signaling was activated in the nascent HSCs at both sites. In the P-Sp explant culture, the overexpression of Dll1 in OP9 stromal cells rescued the failed production of hematopoietic progenitors in the Mib1(-/-) P-Sp, while its activity was abolished by Mib1 knockdown. These results suggest that Mib1 is important for intraembryonic hematopoiesis not only in the aortic endothelium but also in the SAPs.

Escherichia coli enzyme IIANtr regulates the K+ transporter TrkA.

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K(+), cytoplasmic K(+) in bacteria is required for diverse processes. Here, we show that enzyme IIA(Ntr) (EIIA(Ntr)) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K(+) transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIA(Ntr) was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIA(Ntr) for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K(+) level in culture media is lowered. We demonstrate that dephosphorylated EIIA(Ntr), but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K(+). High cellular K(+) levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIA(Glc) regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIA(Ntr) regulates the transport system for K(+), which has global effects related to nitrogen metabolism.

Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells.

Angiopoietin-1 (Ang1) and its receptor, Tie2, play critical roles in blood vessel formation. Ang1 triggers a variety of signaling events in endothelial cells leading to vasculogenic and angiogenic processes. However, the underlying mechanism for Ang1/Tie2 signaling is not fully understood. Here, we show that Tie2 and phospholipase D (PLD) are localized in the caveolae, specialized subdomains of the endothelial cell plasma membrane enriched with signaling molecules. Interestingly, Ang1 increased PLD activities in a dose- and time-dependent manner. Ang1-induced MEK/ERK activation was abrogated when PLD was inhibited, suggesting that PLD mediates Ang1-induced MEK/ERK activation. Moreover, PLD inhibitor, 1-butanol, inhibited Ang1-induced endothelial cell migration. Our results indicate that: (1) caveolae may be the platform for Tie2/PLD association in endothelial cells; (2) PLD is a new mediator of Ang1/Tie2-induced signaling pathway, and it participates in MAPK activation and endothelial cell migration.

Osteoprotegerin ligand induces beta-casein gene expression through the transcription factor CCAAT/enhancer-binding protein beta.

Osteoprotegerin ligand (OPGL, also known as RANKL), a member of the tumor necrosis factor superfamily, is essential for mammary gland development during pregnancy in addition to key roles in the immune system and bone development. Here we show that OPGL induces beta-casein transcription through the CCAAT/enhancer-binding protein beta (C/EBPbeta). In both HC11 cell lines and primary mammary epithelial cells, OPGL stimulation triggers rapid nuclear translocation of C/EBPbeta, which is critical for the expression of the beta-casein gene. Mutation of C/EBbeta binding sites in the beta-casein gene promoter completely abrogated OPGL-induced beta-casein promoter activity. By contrast, OPGL stimulation did not result in STAT5 phosphorylation. In vivo immunohistochemistry studies further demonstrated defective nuclear translocation of C/EBPbeta, but normal STAT5 activation, in OPGL-deficient mice. These data show that OPGL is a critical activator of beta-casein gene expression via the transcription factor C/EBPbeta. Our data provide new insights into the understanding of the molecular events involved in milk protein gene expression.