In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Silent chromatin determines target preference of the Saccharomyces retrotransposon Ty5

The HML and HMR mating loci of Saccharomyces cerevisiae are bound in silent chromatin, which is assembled at the flanking E and I transcriptional silencers. The retrotransposon Ty5 preferentially integrates into regions of silent chromatin, and Ty5 insertions near the HMR-E silencer account for ≈2% of total transposition events. Most Ty5 insertions occur within 800 bp on either side of the autonomously replicating consensus sequence within HMR-E. Ty5 target preference is determined by silent chromatin, because integration near HMR-E is abolished in strains with silencer mutations that alleviate transcriptional repression. The recognition of specific DNA sequences per se does not direct integration, rather, it is the protein complex assembled at the silencers. As demonstrated here for Ty5, recognition of specific chromatin domains may be a general mechanism by which retrotransposons and retroviruses determine integration sites.

Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules

Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro. An RNAP molecule remains at a canonical intrinsic terminator for ≈64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.

Origin and evolutionary relationships of giant Galápagos tortoises

Perhaps the most enduring debate in reptile systematics has involved the giant Galápagos tortoises (Geochelone nigra), whose origins and systematic relationships captivated Charles Darwin and remain unresolved to this day. Here we report a phylogenetic reconstruction based on mitochondrial DNA sequences from Galápagos tortoises and Geochelone from mainland South America and Africa. The closest living relative to the Galápagos tortoise is not among the larger-bodied tortoises of South America but is the relatively small-bodied Geochelone chilensis, or Chaco tortoise. The split between G. chilensis and the Galápagos lineage probably occurred 6 to 12 million years ago, before the origin of the oldest extant Galápagos island. Our data suggest that the four named southern subspecies on the largest island, Isabela, are not distinct genetic units, whereas a genetically distinct northernmost Isabela subspecies is probably the result of a separate colonization. Most unexpectedly, the lone survivor of the abingdoni subspecies from Pinta Island (“Lonesome George”) is very closely related to tortoises from San Cristóbal and Española, the islands farthest from the island of Pinta. To rule out a possible recent transplant of Lonesome George, we sequenced DNA from three tortoises collected on Pinta in 1906. They have sequences identical to Lonesome George, consistent with his being the last survivor of his subspecies. This finding may provide guidance in finding a mate for Lonesome George, who so far has failed to reproduce.

Recurrent invasion and extinction of a selfish gene

Homing endonuclease genes show super-Mendelian inheritance, which allows them to spread in populations even when they are of no benefit to the host organism. To test the idea that regular horizontal transmission is necessary for the long-term persistence of these genes, we surveyed 20 species of yeasts for the ω-homing endonuclease gene and associated group I intron. The status of ω could be categorized into three states (functional, nonfunctional, or absent), and status was not clustered on the host phylogeny. Moreover, the phylogeny of ω differed significantly from that of the host, strong evidence of horizontal transmission. Further analyses indicate that horizontal transmission is more common than transposition, and that it occurs preferentially between closely related species. Parsimony analysis and coalescent theory suggest that there have been 15 horizontal transmission events in the ancestry of our yeast species, through simulations indicate that this value is probably an underestimate. Overall, the data support a cyclical model of invasion, degeneration, and loss, followed by reinvasion, and each of these transitions is estimated to occur about once every 2 million years. The data are thus consistent with the idea that frequent horizontal transmission is necessary for the long-term persistence of homing endonuclease genes, and further, that this requirement limits these genes to organisms with easily accessible germ lines. The data also show that mitochondrial DNA sequences are transferred intact between yeast species; if other genes do not show such high levels of horizontal transmission, it would be due to lack of selection, rather than lack of opportunity.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with α-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Plasmodium falciparum antigenic diversity: Evidence of clonal population structure

Plasmodium falciparum, the agent of malignant malaria, is one of mankind’s most severe scourges. Efforts to develop preventive vaccines or remedial drugs are handicapped by the parasite’s rapid evolution of drug resistance and protective antigens. We examine 25 DNA sequences of the gene coding for the highly polymorphic antigenic circumsporozoite protein. We observe total absence of silent nucleotide variation in the two nonrepeated regions of the gene. We propose that this absence reflects a recent origin (within several thousand years) of the world populations of P. falciparum from a single individual; the amino acid polymorphisms observed in these nonrepeat regions would result from strong natural selection. Analysis of these polymorphisms indicates that: (i) the incidence of recombination events does not increase with nucleotide distance; (ii) the strength of linkage disequilibrium between nucleotides is also independent of distance; and (iii) haplotypes in the two nonrepeat regions are correlated with one another, but not with the central repeat region they span. We propose two hypotheses: (i) variation in the highly polymorphic central repeat region arises by mitotic intragenic recombination, and (ii) the population structure of P. falciparum is clonal—a state of affairs that persists in spite of the necessary stage of physiological sexuality that the parasite must sustain in the mosquito vector to complete its life cycle.

Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)6-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Molecular origin of the mosaic sequence arrangements of higher primate α-globin duplication units

The human adult α-globin locus consists of three pairs of homology blocks (X, Y, and Z) interspersed with three nonhomology blocks (I, II, and III), and three Alu family repeats, Alu1, Alu2, and Alu3. It has been suggested that an ancient primate α-globin-containing unit was ancestral to the X, Y, and Z and the Alu1/Alu2 repeats. However, the evolutionary origin of the three nonhomologous blocks has remained obscure. We have now analyzed the sequence organization of the entire adult α-globin locus of gibbon (Hylobates lar). DNA segments homologous to human block I occur in both duplication units of the gibbon α-globin locus. Detailed interspecies sequence comparisons suggest that nonhomologous blocks I and II, as well as another sequence, IV, were all part of the ancestral α-globin-containing unit prior to its tandem duplication. However, sometime thereafter, block I was deleted from the human α1-globin-containing unit, and block II was also deleted from the α2-globin-containing unit in both human and gibbon. These were probably independent events both mediated by independent illegitimate recombination processes. Interestingly, the end points of these deletions coincide with potential insertion sites of Alu family repeats. These results suggest that the shaping of DNA segments in eukaryotic genomes involved the retroposition of repetitive DNA elements in conjunction with simple DNA recombination processes.

Silencer elements controlling the B29 (Igβ) promoter are neither promoter- nor cell-type-specific

The murine B29 (Igβ) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5′ DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5′ DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5′ segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos, and human B29. These 5′ segments also inhibit transcription by the c-fos promoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5′ of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5′ DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.

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.

Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: Toward a genetic model of meningioma progression

Nineteen benign [World Health Organization (WHO) grade I; MI], 21 atypical (WHO grade II; MII), and 19 anaplastic (WHO grade III; MIII) sporadic meningiomas were screened for chromosomal imbalances by comparative genomic hybridization (CGH). These data were supplemented by molecular genetic analyses of selected chromosomal regions and genes. With increasing malignancy grade, a marked accumulation of genomic aberrations was observed; i.e., the numbers (mean ± SEM) of total alterations detected per tumor were 2.9 ± 0.7 for MI, 9.2 ± 1.2 for MII, and 13.3 ± 1.9 for MIII. The most frequent alteration detected in MI was loss on 22q (58%). In MII, aberrations most commonly identified were losses on 1p (76%), 22q (71%), 14q (43%), 18q (43%), 10 (38%), and 6q (33%), as well as gains on 20q (48%), 12q (43%), 15q (43%), 1q (33%), 9q (33%), and 17q (33%). In MIII, most of these alterations were found at similar frequencies. However, an increase in losses on 6q (53%), 10 (68%), and 14q (63%) was observed. In addition, 32% of MIII demonstrated loss on 9p. Homozygous deletions in the CDKN2A gene at 9p21 were found in 4 of 16 MIII (25%). Highly amplified DNA sequences were mapped to 12q13–q15 by CGH in 1 MII. Southern blot analysis of this tumor revealed amplification of CDK4 and MDM2. By CGH, DNA sequences from 17q were found to be amplified in 1 MII and 8 MIII, involving 17q23 in all cases. Despite the high frequency of chromosomal aberrations in the MII and MIII investigated, none of these tumors showed mutations in exons 5–8 of the TP53 gene. On the basis of the most common aberrations identified in the various malignancy grades, a model for the genomic alterations associated with meningioma progression is proposed.