The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity

The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.     

p57Kip2 regulates actin dynamics by binding and translocating LIM-kinase 1 to the nucleus

p57Kip2 is the only cyclin-dependent kinase (Cdk) inhibitor shown to be essential for mouse embryogenesis. The fact suggests that p57 has a specific role that cannot be compensated by other Cdk inhibitors. LIM-kinase 1 (LIMK-1) is a downstream effector of the Rho family of GTPases that phosphorylates and inactivates an actin depolymerization factor, cofilin, to induce the formation of actin fiber. Here we demonstrate that p57 regulates actin dynamics by binding and translocating LIMK-1 from the cytoplasm into the nucleus, which in turn results in a reorganization of actin fiber. The central region of p57, a unique feature among the Cdk inhibitors, and the N-terminal region of LIMK-1, which contains the LIM domains were essential for the interaction. Expression of p57, but not p27Kip1 or a p57 mutant, with a deletion in the central region was shown to induce marked reorganization of actin filament and a translocation of LIMK-1. Our findings indicate p57 may act as a key regulator in embryogenesis by bearing two distinct functions, the regulation of cell cycle through binding to Cdks and the regulation of actin dynamics through binding to LIMK-1, both of which should be important in developmental procedure.     

p21(waf1) mRNA contains a conserved element in its 3'-untranslated region that is bound by the Elav-like mRNA-stabilizing proteins

The Elav-like proteins are specific mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family (HuD, HuC, and Hel-N1) are required for neuronal differentiation. In this report, using purified HuD protein we have localized a high affinity HuD binding site to a 42-nucleotide region within a U-rich tract in the 3'-untranslated region p21(waf1) mRNA. The binding of HuD to this site is readily displaced by an RNA oligonucleotide encoding the HuD binding site of c-fos. The sequence of this binding site is well conserved in human, mouse, and rat p21(waf1) mRNA. p21(waf1) is an inhibitor of cyclin-dependent kinases and proliferating cell nuclear antigen and induces cell cycle arrest at G1/S, a requisite early step in cell differentiation. The identification of an Elav-like protein binding site in the 3'-untranslated region of p21(waf1) provides a novel link between the induction of differentiation, mRNA stability, and the termination of the cell cycle.     

Excitotoxic brain lesion modifies binding to a USF binding site acting as a negative regulatory element in the Protease nexin-1 promoter

This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.     

Asbestos promotes morphological transformation and elevates expression of a gene family invariably induced by tumor promoters in C3H/10T1/2 cells

The murine proliferin gene family, which has been shown to respond consistently to tumor promoters and other cellular pro-oxidant agents in C3H/10T1/2 cells, was used to monitor responses after treatment of these cell cultures with toxic, pro-oxidant asbestos fibres. Proliferin mRNA levels were increased by amosite, crocidolite or chrysotile asbestos fibres, especially in the presence of fresh serum and at low cell densities. Promotion of morphological transformation was confirmed in two-stage focus formation assays using crocidolite at a fibre density that induced proliferin expression. Asbestos-induced gene expression was inhibited by millimolar levels of N-acetylcysteine (NAC), supporting a linkage between: (i) induced oxidant stress that was sufficient to promote morphological transformation; (ii) induction of proliferin expression. Other anti-oxidant compounds (dithiothreitol and pyrrolidine dithiocarbamate) or enzymes (superoxide dismutase and catalase) did not inhibit induced expression. Non-fibrous powders (titanium dioxide, quartz or silica gel) were also effective inducers of proliferin mRNA accumulation. Latex beads and activated charcoal were effective at higher particle densities, implying that ubiquitous particle-induced surface membrane effects can lead to an NAC-reversible step necessary for proliferin induction. The results showed that asbestos resembled all other promoters of morphological transformation in C3H/10T1/2 cells in that an antioxidant-sensitive induction of the proliferin gene family occurred following treatment.