Thinking way out of the box

l objeto de este trabajo es investigar sobre la arquitectura futurista mediante la figura de Jan Kaplický, el cual realizó planteamientos y arquitecturas que todavía hoy son realmente innovadoras e impactantes, centrando el tema en sus propuestas de vanguardia sobre lo doméstico. La obra elegida para este análisis es la House for a Helicopter Pilot (1979), una arquitectura nómada y cinética en que se habitaría una cápsula modular que puede elevar o bajar sus brazos hidráulicos y luego, una vez cansado el usuario del entorno, podría abandonarlo y encontrar otro lugar salvaje en que situarse. Al ser una obra no construida y utópica, sobre la que no hay apenas documentación, se propone como objetivo indagar sobre esta arquitectura alternativa que hoy en día sigue resultando innovadora, y realizar hipótesis explorando los detalles y objetos que aparecen en la escasa documentación existente, profundizando en las ideas utópicas de Jan Kaplicky

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins—ySKP1, CDC53 (Cullin), and the F-box protein CDC4—that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53ts mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2–hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.

CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4+ T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4+ T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.

Cloning and characterization of the mouse vitamin D receptor promoter

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor. The 1.5-kb 5′-flanking region of the new exon 1 was analyzed and revealed the presence of putative cis-acting elements. Despite the absence of a TATA box, this 5′-flanking region contains several characteristics of a GC-rich promoter including four Sp1 sites present in tandem and two CCAAT boxes. Interestingly, the Sp1 site that is the most proximal to the new exon 1 overlaps a perfect site for Krox-20/24. Krox-20 is a transcription factor involved in brain development, and also in bone remodeling. In luciferase reporter gene expression assays, we showed that sequences from this 5′-flanking region elicit high transactivation activity. Furthermore, in the NIH 3T3 cell line, a 3- to 5-fold increase in response to forskolin treatment (an activator of adenylate cyclase and in turn of protein kinase A pathway) was observed.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein ɛ-deficient mice

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein ɛ (C/EBPɛ) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBPɛ develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3–5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBPɛ is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, Plk, in Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed “polo-box” in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

Pontin52, an interaction partner of β-catenin, binds to the TATA box binding protein

β-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, β-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of β-catenin. Although β-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of β-catenin, a nuclear protein designated Pontin52. Pontin52 binds β-catenin in the region of Armadillo repeats 2–5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, β-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of β-catenin.

Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers

The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPα and p30C/EBPα in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPα levels and binding activity, whereas those of p20C/EBPα and p20C/EBPβ are increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.