Deregulated expression of c-Myc in a translocation-negative plasmacytoma on extrachromosomal elements that carry IgH and myc genes

The induced expression of c-Myc in plasmacytomas in BALB/c mice is regularly associated with nonrandom chromosomal translocations that juxtapose the c-myc gene to one of the Ig loci on chromosome 12 (IgH), 6 (IgK), or 16 (IgL). The DCPC21 plasmacytoma belongs to a small group of plasmacytomas that are unusual in that they appear to be translocation-negative. In this paper, we show the absence of any c-myc-activating chromosomal translocation for the DCPC21 by using fluorescent in situ hybridization, chromosome painting, and spectral karyotyping. We find that DCPC21 harbors c-myc and IgH genes on extrachromosomal elements (EEs) from which c-myc is transcribed, as shown by c-myc mRNA tracks and extrachromosomal gene transfer experiments. The transcriptional activity of these EEs is supported further by the presence of the transcription-associated phosphorylation of histone H3 (H3P) on the EEs. Thus, our data suggest that in this plasmacytoma, c-Myc expression is achieved by an alternative mechanism. The expression of the c-Myc oncoprotein is initiated outside the chromosomal locations of the c-myc gene, i.e., from EEs, which can be considered functional genetic units. Our data also imply that other “translocation-negative” experimental and human tumors with fusion transcripts or oncogenic activation may indeed carry translocation(s), however, in an extrachromosomal form.

The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997) Genes Dev. 11, 345–357]. Although cac1Δ cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual cac1Δ cells switch from transcriptionally “off” to transcriptionally “on” more often per cell cycle than wild-type cells. In addition, cac1Δ cells were defective for transcriptional silencing near internal tracts of C1–3A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription–PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C1–3A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the Escherichia coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.

Differential use of the signal recognition particle translocase targeting pathway for inner membrane protein assembly in Escherichia coli

Assembly of several inner membrane proteins—leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep—has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: The case of HIC-1 and γFBP-B

Hypermethylated in cancer (HIC-1), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five C2H2 zinc fingers and an N-terminal broad complex, tramtrack, and bric à brac/poxviruses and zinc-finger (BTB/POZ) domain found in actin binding proteins or transcriptional regulators involved in chromatin modeling. In the human B cell lymphoma (BCL-6) and promyelocityc leukemia (PLZF) oncoproteins, this domain mediates transcriptional repression through its ability to recruit a silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (N-CoR)-mSin3A-histone deacetylase (HDAC) complex, a mechanism shared with numerous transcription factors. HIC-1 appears unique because it contains a 13-aa insertion acquired late in evolution, because it is not found in its avian homologue, γF1-binding protein isoform B (γFBP-B), a transcriptional repressor of the γF-crystallin gene. This insertion, located in a conserved region involved in the dimerization and scaffolding of the BTB/POZ domain, mainly affects slightly the ability of the HIC-1 and γFBP-B BTB/POZ domains to homo- and heterodimerize in vivo, as shown by mammalian two-hybrid experiments. Both the HIC-1 and γFBP-B BTB/POZ domains behave as autonomous transcriptional repression domains. However, in striking contrast with BCL-6 and PLZF, both HIC-1 and γFBP-B similarly fail to interact with members of the HDAC complexes (SMRT/N-CoR, mSin3A or HDAC-1) in vivo and in vitro. In addition, a general and specific inhibitor of HDACs, trichostatin A, did not alleviate the HIC-1- and γFBP-B-mediated transcriptional repression, as previously shown for BCL-6. Taken together, our studies show that the recruitment onto target promoters of an HDAC complex is not a general property of transcriptional repressors containing a conserved BTB/POZ domain.

The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli

Despite the widely accepted view that transcription of gid and mioC is required for efficient initiation of cloned oriC, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal oriC. Furthermore, neither gid nor mioC transcription is required in cells deficient in the histone-like proteins Fis or IHF. However, oriC that is sufficiently impaired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes. We conclude that transcription of mioC and especially gid is needed to activate oriC only under suboptimal conditions. We suggest that either the rifampicin-sensitive step of initiation is some other transcription occurring from promoter(s) within oriC, or the original inference of transcriptional activation derived from the rifampicin experiments is incorrect.

Archaeal nucleosomes

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect ≈60 bp of DNA and multiples of ≈60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.

The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in Cycling Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3′- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3′-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"–green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.

The Nuclear Actin-related Protein of Saccharomyces cerevisiae, Act3p/Arp4, Interacts with Core Histones

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.

Organization of Highly Acetylated Chromatin around Sites of Heterogeneous Nuclear RNA Accumulation

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into “chromonema” fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi MembranesV⃞

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF4− but was insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF4− were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.

Coilin Can Form a Complex with the U7 Small Nuclear Ribonucleoprotein

Coiled bodies (CBs) in the amphibian oocyte nucleus are spherical structures up to 10 μm or more in diameter, much larger than their somatic counterparts, which rarely exceed 1 μm. Oocyte CBs may have smaller granules attached to their surface or embedded within them, which are identical in structure and composition to the many hundreds of B-snurposomes found free in the nucleoplasm. The matrix of the CBs contains the diagnostic protein p80-coilin, which is colocalized with the U7 small nuclear ribonucleoprotein (snRNP), whereas the attached and embedded B-snurposomes contain splicing snRNPs. A few of the 50–100 CBs in the oocyte nucleus are attached to lampbrush chromosomes at the histone gene loci. By coimmunoprecipitation we show that coilin and the U7 snRNP can form a weak but specific complex in the nucleoplasm, which is dependent on the special U7 Sm-binding site. Under the same conditions coilin does not associate with the U1 and U2 snRNPs. Coilin is a nucleic acid-binding protein, as shown by its interaction with single-stranded DNA and with poly r(U) and poly r(G). We suggest that an important function of coilin is to form a transient complex with the U7 snRNP and accompany it to the CBs. In the case of CBs attached to chromosomes at the histone gene loci, the U7 snRNP is thus brought close to the actual site of histone pre-mRNA transcription.

Regulation of the Anaphase-promoting Complex/Cyclosome by bimAAPC3 and Proteolysis of NIMA

Surprisingly, although highly temperature-sensitive, the bimA1APC3 anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1APC3 is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34cdc2 kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1APC3-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1APC3 double mutant arrests in a mitotic state with very high p34cdc2 H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1APC3 mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1APC3. The bimA1APC3 mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.