Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2ΔC) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2ΔC protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

Rescue of a Wnt mutation by an activated form of LEF-1: Regulation of maintenance but not initiation of Brachyury expression

Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with β-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in Lef1−/−Tcf1−/− mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the β-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation.

A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix.

A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol IIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.

Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.

The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.

A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals.

We describe a conserved family of bacterial gene products that includes the VirB1 virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-host-range bacterial plasmids, and gene products that may be involved in invasion by Shigella spp. and Salmonella enterica. Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold. Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity. Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme. Two acidic residues in the VirB1 protein of Agrobacterium tumefaciens form a putative catalytic dyad, Each of these residues was changed into the corresponding amide by site-directed mutagenesis. Strains of A. tumefaciens that express mutated VirB1 proteins have a significantly reduced virulence. We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave beta-1,4-glycosidic bonds as part of their function.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors.

Human transcription initiation factor TFIID is composed of the TATA-binding polypeptide (TBP) and at least 13 TBP-associated factors (TAFs) that collectively or individually are involved in activator-dependent transcription. To investigate protein-protein interactions involved in TFIID assembly and in TAF-mediated activator functions, we have cloned and expressed cDNAs encoding human TAFII80 and TAFII31. Coimmunoprecipitation assays showed that TAFII80 interacted with TAFII250, TAFII31, TAFII20, and TBP, but not with TAFII55. Similar assays showed that TAFII80 interacted with TFIIE alpha and with TFIIF alpha (RAP74) but not with TFIIB, TFIIE beta, or TFIIF beta (RAP30). Further studies with TAFII80 mutations revealed three distinct interaction domains which fall within regions conserved in human TAFII80, Drosophila TAFII60, and yeast TAFII60. The N terminus of TAFII80 (residues 1-100) interacts with both TAFII31 and TAFII20, while two C-terminal regions are involved, respectively, in interactions with TAFII250 and TFIIF alpha (RAP74) (residues 203-276) and with TBP and TFIIE alpha (residues 377-505). The interactions between TAFII80 and general factors TFIIE alpha and TFIIF alpha (RAP74) could be important for recruitment of GTFs during activator-dependent transcription. Because TAFs 80, 31, and 20 show sequence similarities to histones H4, H3, and H2B, as well as some parallel interactions, this subset of TAFs may form a related core structure within TFIID.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Seasonal development and yield of native plants on the upper Snake River Plains and their relation to certain climatic factors /

Issued also in microfilm form, as thesis, University of Minnesota, under title: Seasonal development and yield of native plants on the upper Snake River Plains of Idaho and their relation to climatic factors, especially precipitation and temperature.

Frequency, Form, and Distribution of Illocutionary Speech Acts in Swedish Parent-Child Interaction

In this study, young children’s development of speech acts was examined. Interaction between six Swedish-speaking parents and their children was observed. The frequency, form and distribution of speech acts in the output from the parents were compared with the frequency, form and distribution of the children’s speech acts. The frequency was measured by occurrences per analysed session. The aim of the analysis was to examine if the parent’s behaviour could be treated as a baseline for the child’s development. Both the parents’ and the children’s illocutionary speech acts were classified. Each parent-child dyad was observed at four different occasions, when the children were 1;0, 1;6, 2;0, and 2;6 years of age. Similar studies have previously shown that parents keep a consistent frequency of speech acts within a given time span of interaction, though the distribution of different types of speech acts may shift, depending on contextual factors. The form, in terms of Mean Length of Speech Act in Words (MLSAw), were correlated with the longitudinal result of the children’s MLSAw. The distribution of the parents’ speech acts showed extensive individual differences. The result showed that the children’s MLSAw move significantly closer the MLSAw of their parents. Since the parent’s MLSAw showed a wide distribution, these results indicate that the parent’s speech acts can be treated as a baseline for certain aspects of the children’s development, though further studies are needed.

Socio-Historical Factors Mediating Collaborative Teaching and Learning: A Design-Based Investigation and Intervention

Thesis (Ph.D.)--University of Washington, 2016-06

Factors affecting the formation of a skin reservoir for topically applied solutes

The reservoir function of the skin is an important determinant of the duration of action of a topical solute. The reservoir can exist in the stratum corneum, in the viable avascular tissue (viable epidermis and supracapillary dermis) and in the dermis. A steroid reservoir in the stratum corneum has been demonstrated by the reactivation of a vasoconstrictor effect by occlusion or application of a placebo cream to the skin some time after the original topical application of steroid. Other solutes have also been reported to show a reservoir effect in the skin after topical application. A simple compartmental model is used to understand why reactivation of vasoconstriction some time after a topical steroid application shows dependency on time, topical solute concentration and the product used to cause reactivation. The model is also used to show which solutes are likely to show a reservoir effect and could be potentially affected by desquamation, especially when the turnover of the skin is abnormally rapid. A similar form of the model can be used to understand the promotion of reservoir function in the viable tissue and in the dermis in terms of effective removal by blood perfusing the tissues. Copyright (C) 2004 S. Karger AG, Basel.