Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex.

Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TTAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TTAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix-associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.

Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail and found that the interaction is saturable, of high affinity, and evolutionarily conserved. Competition studies revealed the existence of two different types of interaction related to different structural features of MARs: one involving the minor groove of double-stranded MAR DNA and one involving single-stranded regions. We obtained similar results for the interaction of MARs with intact nuclear matrices from rat liver. A model in which the interaction of nuclear matrix proteins with single-stranded MAR regions serves to stabilize the transcriptionally active state of chromatin is discussed.

Interaction between the chemotactic cAMP receptor and a detergent-insoluble membrane residue of Dictyostelium discoideum. Modulation by guanine nucleotides.

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.

Changes in cyclic AMP receptor properties during adaptation in Dictyostelium discoideum.

In developing Dictyostelium discoideum cells, binding of cyclic AMP to the chemotactic receptor has been shown to oscillate. These oscillations represent cycles of activation, adaptation and deadaptation of the cyclic AMP receptor system. We studied the molecular basis of these oscillatory changes in cyclic AMP receptor binding. We developed a rapid method of lysing cells during the course of the oscillations. This method guaranteed good preservation of ligand binding properties of the cyclic AMP receptor. We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed that both GTP and GDP abolished oscillations in receptor binding completely, presumably by acting via a G protein. The guanine nucleotides reduced the affinity of the receptor at all time-points of the oscillation cycle to the minimal, i.e. adapted, level. We conclude that the cyclic process of activation, adaptation and de-adaptation in D. discoideum, at cyclic AMP receptor level, involves changes in receptor-G protein interaction. During adaptation, the affinity of the cyclic AMP receptor decreases and the receptor becomes insensitive to guanine nucleotides.

Identification and analysis of a matrix-attachment region 5' of the rat glutamate-dehydrogenase-encoding gene.

Eukaryotic chromatin is thought to be organized into independently regulated loop domains by interaction of matrix-attachment regions (MAR) of the DNA to the nuclear matrix. To define the borders of the chromatin loop containing the glutamate dehydrogenase (GDH) gene, we screened the GDH gene and flanking regions for the presence of MAR sequences. We here report identification, mapping and sequencing of an (A + T)-rich MAR located 2010-1397 bp upstream of the transcription initiation site of GDH, that mediates strong binding to the nuclear matrix. Smaller regions can also confer binding capacity, although at a lower affinity. This (A + T)-rich MAR contained 11 bp and 12 bp (A + T)-rich direct repeats, but not any of the sequences previously described to be associated with MAR activity. We here show that the presence of (A + T)-rich domains of DNA is not sufficient to confer binding capacity, since (A + T)-rich sequences located downstream of the identified MAR did not bind to the nuclear matrix. Moreover, a consensus topoisomerase-II-binding site located downstream of the MAR was found to be insufficient to mediate substantial binding. The number of binding sites in the nuclear matrix for MAR-containing fragments was shown to be approximately 15,000/nucleus. Since organization of the entire rat genome in loops with an average loop size of 100 kbp would require 60,000 binding sites, this suggests that only part of the genome is organized in loops. Alternatively, we might have underestimated the number of binding sites. The GDH MAR, and MAR-containing fragments derived from other species, were found to bind to the same binding sites in the nuclear matrix, although the affinity varied.

Expression of a mutated ras gene in Dictyostelium discoideum alters the binding of cyclic AMP to its chemotactic receptor.

Dictyostelium discoideum cells contain a ras gene that codes for a polypeptide that is highly homologous to the human ras proteins. Extra copies of the wild-type gene or a gene carrying a missense mutation in codon 12 (ras-Gly12 and ras-Thr12, respectively) have been introduced into Dictyostelium cells by transformation. We have investigated the properties of the chemotactic cell surface cyclic AMP receptor in crude membrane preparations of wild-type Dictyostelium cells and ras-Gly12 and ras-Thr12 transformants. In vitro, an ATP- and Ca2+-dependent reduction of the number of cyclic AMP receptors was observed in membranes from all three cell types. The number of available receptors was decreased maximally by about 50%. In the presence of ATP the half-maximal Ca2+ concentration required for this process was about 10(-5) M in wild-type and ras-Gly12 membranes, and less than 10(-7) M in ras-Thr12 membranes. Addition of GTP (but not GDP) or the phorbol ester PMA (phorbol-12-myristate-13-acetate) reduced the Ca2+ requirement of the process in wild-type and ras-Gly12 membranes to the physiological level of less than 10(-7) M. In membranes derived from ras-Thr12 cells addition of GTP or PMA had no effect. The results indicate that D. discoideum cells contain a cyclic AMP receptor-controlling pathway that can be activated in vitro and involves a GTP-binding protein and a Ca2+ plus ATP-dependent activity, possibly protein kinase C. It is concluded that the ras protein specifically interacts with this pathway; the pathway appears to be constitutively activated by the mutated ras gene product.

Ligand-independent reduction of cAMP receptors in Dictyostelium discoideum cells over-expressing a mutated ras gene.

Drug-resistance selection in Dictyostelium discoideum transformants resulted in up to eight-times-higher ras protein levels. Over-production of the wild-type ras protein did not lead to an aberrant phenotype. Increased levels of the mutated [G12T]ras protein, however, were correlated with severe deficiencies in aggregation and development. This aberrant phenotype is associated with reduced cAMP binding, due to a lower number of cell-surface receptors. We show that both RNA and cAMP-receptor-protein levels are reduced. These results indicate that ras in Dictyostelium discoideum seems to be involved in regulating cAMP-receptor-gene expression.

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.

Mutant ras gene induces elevated levels of inositol tris- and hexakisphosphates in Dictyostelium.

Previous studies of Europe-Finner & Newell indicated that in amoebae of Dictyostelium discoideum, signal transduction used for chemotaxis to cyclic AMP involved transient formation of inositol tris- and polyphosphates. Evidence was also presented for the involvement of a GTP-binding G-protein. Here we report evidence for the involvement of a ras gene product in the D. discoideum inositol phosphate pathway. Use was made of strains of Dictyostelium transformed with a wild-type D. discoideum ras gene (ras-Gly12) or a mutant form of the gene (ras-Thr12). Experiments using separation of soluble inositol phosphates by Dowex anion-exchange resin chromatography indicated that cells transformed with the wild-type ras-Gly12 gene were unaffected in their basal levels of inositol polyphosphates and in the inositol phosphates formed in response to stimulation with the chemotactic agent cyclic AMP. In contrast, cells transformed with the mutant ras-Thr12 gene showed a basal level of inositol polyphosphate that was several-fold elevated over the controls and stimulation of these cells with cyclic AMP produced only a small further elevation. When the inositol phosphates were analysed by h.p.l.c. it was found that the basal level of inositol 1,4,5-trisphosphate was raised three- to fivefold in the ras-Thr12 strain compared to the strain transformed with ras-Gly12, and that inositol hexakisphosphate (which was found to be present in large amounts relative to other inositol phosphates in D. discoideum cells) was also raised to a similar extent in the ras-Thr12-transformed cells. We propose that the Dictyostelium ras gene product codes for a regulatory protein involved in the inositol phosphate chemotactic signal-transduction pathway.