A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and β-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the α-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, β-galactosidase and immunochemical stainings revealed differences in GFP and β-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense β-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Clustering of meiotic double-strand breaks on yeast chromosome III

In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-strand breaks (DSBs) that are repaired by interaction of the broken chromosome with its homologue. To identify a large number of DSB sites and gain insight into the control of DSB formation at both the local and the whole chromosomal levels, we have determined at high resolution the distribution of meiotic DSBs along the 340 kb of chromosome III. We have found 76 DSB regions, mostly located in intergenic promoter-containing intervals. The frequency of DSBs varies at least 50-fold from one region to another. The global distribution of DSB regions along chromosome III is nonrandom, defining large (39–105 kb) chromosomal domains, both hot and cold. The distribution of these localized DSBs indicates that they are likely to initiate most crossovers along chromosome III, but some discrepancies remain to be explained.

An HP1-like protein is missing from transcriptionally silent micronuclei of Tetrahymena

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.

Repetitive-DNA elements are similarly distributed on Caenorhabditis elegans autosomes

The positions of ≈4,800 individual miniature inverted-repeat transposable element (MITE)-like repeats from four families were mapped on the Caenorhabditis elegans chromosomes. These families represent 1–2% of the total sequence of the organism. The four MITE families (Cele1, Cele2, Cele14, and Cele42) displayed distinct chromosomal distribution profiles. For example, the Cele14 MITEs were observed clustering near the ends of the autosomes. In contrast, the Cele2 MITEs displayed an even distribution through the central autosome domains, with no evidence for clustering at the ends. Both the number of elements and the distribution patterns of each family were conserved on all five C. elegans autosomes. The distribution profiles indicate chromosomal polarity and suggest that the current genetic and physical maps of chromosomes II, III, and X are inverted with respect to the other chromosomes. The degree of conservation of both the number and distribution of these elements on the five autosomes suggests a role in defining specific chromosomal domains.

A cis-acting element that directs the activity of the murine methylation modifier locus Ssm1

Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature of Ssm1’s targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism of Ssm1 action are discussed.

Sir3-dependent assembly of supramolecular chromatin structures in vitro

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.

Cell Cycle–regulated Transcription in Fission Yeast: Cdc10–Res Protein Interactions during the Cell Cycle and Domains Required for Regulated Transcription

In Schizosaccharomyces pombe the MBF (DSC1) complex mediates transcriptional activation at Start and is composed of a common subunit called Cdc10 in combination with two alternative DNA-binding partners, Res1 and Res2. It has been suggested that a high-activity MBF complex (at G1/S) is switched to a low-activity complex (in G2) by the incorporation of the negative regulatory subunit Res2. We have analyzed MBF protein–protein interactions and find that both Res proteins are associated with Cdc10 throughout the cell cycle, arguing against this model. Furthermore we demonstrate that Res2 is capable of interacting with a mutant form of Cdc10 that has high transcriptional activity. It has been shown previously that both Res proteins are required for periodic cell cycle–regulated transcription. Therefore a series of Res1–Res2 hybrid molecules was used to determine the domains that are specifically required to regulate periodic transcription. In Res2 the nature of the C-terminal region is critical, and in both Res1 and Res2, a domain overlapping the N-terminal ankyrin repeat and a recently identified activation domain is important for mediating cell cycle–regulated transcription.

An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death.

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.

Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

Natural genetic exchange between Haemophilus and Neisseria: Intergeneric transfer of chromosomal genes between major human pathogens

Members of the bacterial families Haemophilus and Neisseria, important human pathogens that commonly colonize the nasopharynx, are naturally competent for DNA uptake from their environment. In each genus this process is discriminant in favor of its own and against foreign DNA through sequence specificity of DNA receptors. The Haemophilus DNA uptake apparatus binds a 29-bp oligonucleotide domain containing a highly conserved 9-bp core sequence, whereas the neisserial apparatus binds a 10-bp motif. Each motif (“uptake sequence”, US) is highly over-represented in the chromosome of the corresponding genus, particularly concentrated with core sequences in inverted pairs forming gene terminators. Two Haemophilus core USs were unexpectedly found forming the terminator of sodC in Neisseria meningitidis (meningococcus), and sequence analysis strongly suggests that this virulence gene, located next to IS1106, arose through horizontal transfer from Haemophilus. By using USs as search strings in a computer-based analysis of genome sequence, it was established that while USs of the “wrong” genus do not occur commonly in Neisseria or Haemophilus, where they do they are highly likely to flag domains of chromosomal DNA that have been transferred from Haemophilus. Three independent domains of Haemophilus-like DNA were found in the meningococcal chromosome, associated respectively with the virulence gene sodC, the bio gene cluster, and an unidentified orf. This report identifies intergenerically transferred DNA and its source in bacteria, and further identifies transformation with heterologous chromosomal DNA as a way of establishing potentially important chromosomal mosaicism in these pathogenic bacteria.

SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor α (RARα) and PLZF-RARα oncoproteins associated with acute promyelocytic leukemia

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARα. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARα and PLZF-RARα oncoproteins retain the ability of RARα to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors.

Characterization of the CHD family of proteins

The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that bound a DNA promoter element. The presence of chromo (chromatin organization modifier) domains and an SNF2-related helicase/ATPase domain led to speculation that this gene regulated chromatin structure or gene transcription. This study describes the cloning and characterization of three novel human genes related to MmCHD1. Examination of sequence databases produced several more related genes, most of which were not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. The major region of sequence variation is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggested that enhanced transcriptional arrest at RNA polymerase II pause sites due to 6-azauracil-induced nucleotide pool depletion was reduced in the deletion strain and that ScCHD1 inhibited transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template.

HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1–56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.