Biomedical subjects
Publications and source records attributed to G R Crabtree.
Histone acetyltransferases and deacetylases are involved in the regulation of gene transcription. Recently, tumor suppressor protein p53 has been shown to be a target for transcriptional coactivators that have histone acetyltransferase activity, suggesting acetylation is also involved in the regulation of cell proliferation and tumorigenesis.
BACKGROUND: Understanding the cellular role of a protein often requires a means of altering its function, most commonly by mutating the gene encoding the protein. Alternatively, protein function can be altered directly using a small molecule that binds to the protein, but no general method exists for the systematic discovery of small molecule ligands. Split-pool synthesis provides a means of synthesizing vast numbers of small molecules. Synthetic chemists will soon be able to synthesize natural product-like substances by this method, so compatible screening methods that detect the activity of minute quantities of molecules among many inactive ones will be in demand. RESULTS: We describe two advances towards achieving the above goals. First, a technique is described that uses a simple spray gun to create 5000-8000 droplets randomly, each having a volume of 50-200 nanoliters. The individual 'nanodroplets' contain a controlled number of cells and many also contain individual synthesis beads. As small molecules can be photochemically released from the beads in a time-dependent manner, the concentration of ligands that the cells are exposed to can be controlled. The spatial segregation of nanodroplets prevents the mixing of compounds from other beads so the effects of each molecule can be assayed individually. Second, a small molecule-dependent genetic selection involving engineered budding yeast cells was used to detect intracellular protein-ligand interactions in nanodroplets. CONCLUSIONS: The technique described here should facilitate the discovery of new cell-permeable ligands, especially when combined with a positive selection assay that detects intracellular binding of small molecules to proteins. Using 'anchored combinatorial libraries', it may be possible to screen entire libraries of natural product-like molecules against the entire collection of proteins encoded within cDNA libraries in a single experiment.
It is not known how immunogenic versus tolerogenic cellular responses are signaled by receptors such as the B cell antigen receptor (BCR). Here we compare BCR signaling in naive cells that respond positively to foreign antigen and self-tolerant cells that respond negatively to self-antigen. In naive cells, foreign antigen triggered a large biphasic calcium response and activated nuclear signals through NF-AT, NF-kappa B, JNK, and ERK/pp90rsk. In tolerant B cells, self-antigen stimulated low calcium oscillations and activated NF-AT and ERK/pp90rsk but not NF-kappa B or JNK. Self-reactive B cells lacking the phosphatase CD45 did not exhibit calcium oscillations or ERK/pp90rsk activation, nor did they repond negatively to self-antigen. These data reveal striking biochemical differences in BCR signaling to the nucleus during positive selection by foreign antigens and negative selection by self-antigens.
The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.
Although shown to be highly expressed by the epidermis in inflammatory skin disease, the ability of the Fas protein to trigger apoptosis in the distinct cell subpopulations of cutaneous tissue, particularly with regard to receptor density and the degree of crosslinking, has not been fully characterized. We therefore determined the effect of Fas cross-linking in primary human dermal fibroblasts at both high and low levels of Fas receptor expression. First, we examined the effects of the anti-Fas monoclonal antibody, CH-11, on fibroblasts expressing low basal levels of Fas. In these cells Fas aggregation stimulated proliferation by 160 +/- 10% over untreated controls. In contrast, the same concentration of CH-11 had an inhibitory effect on epidermal keratinocyte growth. Because Fas is upregulated in inflamed skin, we next examined the effects of Fas cross-linking on fibroblasts expressing augmented levels ofFas. Fibroblasts were either transfected with plasmids for overexpression of full length or bioengineered Fas receptors or were transduced with a retroviral Fas expression vector. In these cells Fas oligomerization triggered the morphologic changes indicative of apoptosis regardless of whether or not the Fas-signaling domain was tethered to the plasma membrane. These studies indicate that Fas oligomerization in dermal fibroblasts may initiate dual signaling programs, either proliferation or apoptosis, and that the chosen outcome may depend upon the magnitude of Fas aggregation.
The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.
Cells need to distinguish between transient Ca2+ signals that induce events such as muscle contraction, secretion, adhesion and synaptic transmission, and sustained Ca2+ signals that are involved in cell proliferation and differentiation. The latter class of events is blocked in lymphocytes by the immunosuppressive drugs cyclosporin A and FK506, which inhibit calcineurin, a Ca2+-activated serine/threonine phosphatase necessary for the nuclear import of NF-AT transcription factors. Here we report that sustained high concentrations of Ca2+, but not transient pulses, are required to maintain NF-AT transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation. Furthermore, overexpression and constitutive nuclear localization of NF-AT, but not Jun, Fos, NF-kappaB, Oct or Ets family members, renders the interleukin-2 enhancer in Jurkat T lymphocytes resistant to FK506 and cyclosporin A. Thus a primary effect of these immunosuppressive reagents is to control the subcellular localization of the NF-AT family of transcription factors.
We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.
The SWI/SNF complex in yeast facilitates the function of transcriptional activators by opposing chromatin-dependent repression of transcription. We demonstrate that in mammals SWI/SNF complexes are present in multiple forms made up of 9-12 proteins that we refer to as BRG1-associated factors (BAFs) ranging from 47 to 250 kD. We have isolated cDNAs for human BAF155, BAF170, and BAF60. BAF155 and BAF170 are encoded by separate genes that are both homologs of yeast SWI3. Both contain a region of similarity to the DNA binding domain of myb, but lack the basic residues known to be necessary for interaction with DNA. The two SWI3 homologs copurify on antibody columns specific for either BAF155 or BAF170, indicating that they are in the same complex. BAF60 is encoded by a novel gene family. An open reading frame from yeast, which is highly homologous, encodes the previously uncharacterized 73-kD subunit of the yeast SWI/SNF complex required for transcriptional activation by the glucocorticoid receptor (Cairns et al., this issue). BAF60a is expressed in all tissues examined, whereas BAF60b and BAF60c are expressed preferentially in muscle and pancreas, respectively. BAF60a is present within the 2000-kD BRG1 complex, whereas BAF60b is in a distinct complex that shares some but not all subunits with the BRG1 complex. The observed similarity between mammalian BAF190, BAF170, BAF155, BAF60, and BAF47 and yeast SNF2/SWI2, SWI3, SWI3, SWP73, and SNF5, respectively, underscores the similarity of the mammalian and yeast complexes. However, the complexes in mammals are more diverse than the SWI/SNF complex in yeast and are likely dedicated to developmentally distinct functions.
Eukaryotic transcriptional activators mediate transcriptional induction through stabilization of the preinitiation complex, probably through direct interactions with basal transcription factors. In vitro studies on the role of an activator in the maintenance of on-going transcription (reinitiation) have been contradictory, suggesting that, after formation of a preinitiation complex, an activator may or may not be necessary for transcription to be maintained. We have developed a means of regulating transcription in living cells through the use of both homodimeric and heterodimerizing synthetic ligands that allow the ligand-dependent association and disassociation of a transcriptional activation domain with a promoter. Here we report that maintaining the transcription of endogenous genes in vivo, in both yeast and human cells, requires the continuous presence of the activation domain. The use of synthetic ligands as a transcriptional on-off switch represents a powerful means of controlling the transcription in vitro and in vivo for both experimental and therapeutic purposes.
BACKGROUND: Genetic abnormalities in the Fas receptor or its trimeric ligand, FasL, result in massive T-cell proliferation and a lupus-like autoimmune syndrome, which was initially attributed to excessive lymphoproliferation but is now ascribed to the absence of Fas-mediated cell death. Although Fas is normally expressed on most thymocytes, negative selection seems to be unperturbed in Fas-deficient (lpr) mice. This suggests that Fas has an important function in peripheral, but not thymic, T cells. RESULTS: To explore the Fas-mediated cell death pathway both in vitro and in vivo, we used conditional alleles of the Fas receptor that can be triggered by an intracellularly active chemical inducer of dimerization known as FK1012. We found that membrane attachment is important for Fas function and, unlike previous results with anti-Fas monoclonal antibodies, we show that dimerization is sufficient to trigger apoptosis. Finally, the administration of FK1012 in vivo to transgenic animals expressing the conditional FAS receptor in thymocytes demonstrates that sensitivity to FAS-mediated apoptosis is restricted to CD4+CD8+ thymocytes. CONCLUSIONS: Here, we describe the first in vivo application of non-toxic, cell-permeable synthetic ligands to regulate signal transduction in transgenic mice expressing a conditional receptor. Using this system, we show that the Fas pathway is restricted to double-positive thymocytes in vivo, consistent with recent in vitro findings with thymocytes. This method promises to be more useful not only for developmental studies involving cell ablation, but also for studies involving the regulation of a wide variety of signaling molecules.
Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo- or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. The ability to induce signals at each of these three phases of signaling pathways is illustrated by the use of a heterodimeric chemical inducer of dimerization that causes a proximal relationship between two different target proteins.
The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin suppress the immune response by inhibiting evolutionary conserved signal transduction pathways. CsA, FK506, and rapamycin bind to their intracellular receptors, immunophilins, creating composite surfaces that block the activity of specific targets. For CsA/cyclophilin and FK506/FKBP the target is calcineurin. Because of the large surface area of interaction of the drug-immunophilin complex with calcineurin, FK506 and CsA have a specificity for their biologic targets that is equivalent to growth factor-receptor interactions. To date, all the therapeutic as well as toxic effects of these drugs have been shown to be due to inhibition of calcineurin. Inhibition of the action of calcineurin results in a complete block in the translocation of the cytosolic component of the nuclear factor of activated T cells (NF-AT), resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin (IL-4) and CD40 ligand as well as those necessary for T-cell proliferation such as IL-2. The purpose of this article is to illustrate the means by which these drugs produce immunosuppression.
Everyone has had the experience of stoking a fire; putting the logs close together causes flames to appear from previously dying embers. In a similar way, mere proximity might mediate qualitative biological responses. We discuss natural molecules that appear to have arisen to bring two proteins together and illustrate how this simple mechanism can be used to control a wide variety of biological processes.
BACKGROUND: Cell death can occur either from physical damage (necrosis) or cellular suicide (apoptosis). Apoptosis is essential for the development of multicellular organisms and disregulated apoptosis underlies many human diseases. The Fas receptor (Fas) is a membrane signaling protein that mediates a death signal following its aggregation by the Fas ligand. We have described methods to induce the association of proteins using cell-permeable molecules called chemical inducers of dimerization (CIDs). Here we describe the synthesis of a novel CID, (CsA)2, that has two identical protein-binding surfaces derived from the immunosuppressant cyclosporin A (CsA). We use this CID to deliver a death signal to cells expressing a fusion protein containing cyclophilin (CyP, the protein receptor for cyclosporin) and the cytoplasmic signaling domain of Fas. RESULTS: (CsA)2 was synthesized in six synthetic steps and 30% overall yield from cyclosporin. It binds to two CyP proteins simultaneously, but does not inhibit T-cell signaling, presumably because the (CsA)2-CyP complex does not bind to calcineurin. Jurkat cells stably transfected with constructs encoding myristoylated CyP-Fas fusion proteins undergo apoptosis in response to nanomolar quantities of (CsA)2. Constructs containing a mutation in the myristoylation signal are defective for signaling. CONCLUSIONS: The Fas signaling pathway can be activated with a cell-permeable CID derived from CsA in cells expressing an appropriately engineered Fas construct, which must be localized at the membrane. This new class of homodimerizing CIDs will be useful for in-depth analysis of protein association events in complex systems, including transgenic animals. Now that several CIDs with distinct dimerization characteristics are available, it should be possible to induce the activation of multiple pathways with complete specificity.
The Src-like tyrosine kinases require membrane localization for transformation and probably for their normal role in signal transduction. We utilized this characteristic to prepare Src-like tyrosine kinases that can be readily activated with the rationally designed chemical inducer of dimerization FK1012. Dimerization of cytoplasmic Src-like tyrosine kinases was not sufficient for signaling, but their recruitment to the plasma membrane led to the rapid activation of transcription factors identical to those regulated by crosslinking the antigen receptor. Moreover, recruitment of activated Src-like kinases to the membrane replaced signaling by the T-lymphocyte antigen receptor complex, leading to the activation of both the Ras/protein kinase C and Ca2+/calcineurin pathways normally activated by antigen receptor signaling. Since these chemical inducers of dimerization are cell permeable, this approach permits the production of conditional alleles of any of the Src-like tyrosine kinases, thereby allowing a delineation of their developmental roles.
While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Activation of Sos appears to involve membrane recruitment and not allosteric changes, because interaction of Sos with the linking molecule Grb-2 was not required for Ras activation. To extend this analysis, we constructed a modified Sos that could be localized to the membrane inducibly by using a rationally designed chemical inducer of dimerization, FK1012. The role of Grb-2 in signaling was mimicked with this technique, which induced the association of a modified Sos with the membrane, resulting in rapid activation of Ras-induced signaling. In contrast, inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation. This conditional allele of Sos demonstrates that membrane localization of Sos is sufficient for Ras activation in T cells and indicates that the role of Grb-2 is to realize the biologic advantages of linker-mediated dimerization: enhanced specificity and favorable kinetics for signaling. This method of generating conditional alleles may also be useful in dissecting other signal transduction pathways regulated by protein localization or protein-protein interactions.
Signals transduced by the T cell antigen receptor (TCR) regulate developmental transitions in the thymus and also mediate the immunologic activation of mature, peripheral T cells. In both cases TCR stimulation leads to the assembly of the NFAT transcription complex as a result of the calcium-dependent nuclear translocation of cytosolic subunits, NFATc, and the Ras/protein kinase C-dependent induction of a nuclear subunit, NFATn. To further understand the diverse roles of antigen receptor signaling throughout T cell development, we have identified a new NFATc family member, NFATc3, that is expressed at highest levels in the thymus. NFATc3 is the product of a gene on murine chromosome 8 that is not linked to the other NFATc genes. NFATc3, like other NFATc family members, contains a conserved rel similarity domain, and also defines a region conserved among NFATc family members, the SP repeat region, characterized by the repeated motif SPxxSPxxSPrxsxx (D/E)(D/E)swl. NFATc3 activates NFAT site-dependent transcription when overexpressed, yet exhibits a pattern of DNA site specificity distinct from other NFATc proteins. Additionally, thymic NFATc3 undergoes modifications in response to agents that mimic T cell receptor signaling, including a decrease in apparent molecular mass upon elevation of intracellular calcium that is inhibited by the immunosuppressant FK506. Given the preferential expression of NFATc3 in the thymus, NFATc family members may regulate distinct subsets of genes during T cell development.