Biomedical subjects

Edward M Rubin

Publications and source records attributed to Edward M Rubin.

At least 37 records · Page 2Linked to original sources

PubMed2005

In vivo characterization of a vertebrate ultraconserved enhancer.

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.

Animals↗

PubMed2005

Comparative genomic analysis reveals a distant liver enhancer upstream of the COUP-TFII gene.

COUP-TFII is a central nuclear hormone receptor that tightly regulates the expression of numerous target lipid metabolism genes in vertebrates. However, it remains unclear how COUP-TFII itself is transcriptionally controlled since studies with its promoter and upstream region fail to recapitulate the gene's liver expression. In an attempt to identify liver enhancers in the vicinity of COUP-TFII, we employed a comparative genomic approach. Initial comparisons between humans and mice of the 3470-kb gene-poor region surrounding COUP-TFII revealed 2023 conserved noncoding elements. To prioritize a subset of these elements for functional studies, we performed further genomic comparisons with the orthologous pufferfish (Fugu rubripes) locus and uncovered two anciently conserved noncoding sequences (CNS) upstream of COUP-TFII (CNS-62kb and CNS-66kb). Testing these two elements using reporter constructs in liver cells (HepG2) revealed that CNS-66kb, but not CNS-62kb, yielded robust in vitro enhancer activity. In addition, an in vivo reporter assay using naked DNA transfer with CNS-66kb linked to luciferase displayed strong reproducible liver expression in adult mice, further supporting its role as a liver enhancer. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the substrates to better understand the regulation and expression of COUP-TFII.

Animals↗

PubMed2005

Metagenomics: DNA sequencing of environmental samples.

Although genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent only a fraction of the living or once-living organisms of interest. Many species are difficult to study in isolation because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. Methods that are based on DNA sequencing circumvent these obstacles, as DNA can be isolated directly from living or dead cells in various contexts. Such methods have led to the emergence of a new field, which is referred to as metagenomics.

Animals↗

PubMed2005

Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor.

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.

Adult↗

PubMed2004

Mouse strain-specific differences in vascular wall gene expression and their relationship to vascular disease.

OBJECTIVE: Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice. METHODS AND RESULTS: We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis. CONCLUSIONS: Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study. To gain insights into the molecular pathways that are differentially activated in strains of mice with varied susceptibility to atherosclerosis, we performed comprehensive transcriptional profiling of their vascular wall. Genes identified through these studies expand the repertoire of factors in disease-related signaling pathways and identify novel candidate genes in atherosclerosis.

Aging↗

PubMed2004

Intraspecies sequence comparisons for annotating genomes.

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intraspecies sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents, and a set of genomic intervals were amplified, resequenced, and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C. intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom. It also raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species.

Animals↗

PubMed2004

Megabase deletions of gene deserts result in viable mice.

The functional importance of the roughly 98% of mammalian genomes not corresponding to protein coding sequences remains largely undetermined. Here we show that some large-scale deletions of the non-coding DNA referred to as gene deserts can be well tolerated by an organism. We deleted two large non-coding intervals, 1,511 kilobases and 845 kilobases in length, from the mouse genome. Viable mice homozygous for the deletions were generated and were indistinguishable from wild-type littermates with regard to morphology, reproductive fitness, growth, longevity and a variety of parameters assaying general homeostasis. Further detailed analysis of the expression of multiple genes bracketing the deletions revealed only minor expression differences in homozygous deletion and wild-type mice. Together, the two deleted segments harbour 1,243 non-coding sequences conserved between humans and rodents (more than 100 base pairs, 70% identity). Some of the deleted sequences might encode for functions unidentified in our screen; nonetheless, these studies further support the existence of potentially 'disposable DNA' in the genomes of mammals.

Animals↗

PubMed2004

Exploiting human--fish genome comparisons for deciphering gene regulation.

Comparative genomics has served as an essential guide in the identification of functional coding and non-coding sequences in vertebrate genomes. Human-mouse pair-wise comparisons have limited utility for identifying functional conserved non-coding sequences, owing to the large number of sequences shared between these species. In searching for more stringent filters to uncover non-coding elements more likely to be of functional importance in the human genome, human-fish sequence comparisons have emerged as an important strategy, leading to the efficient identification of enhancer elements. These sequences are unevenly distributed in the genome, tending to cluster around genes involved in key developmental processes, with recent studies suggesting that they represent genomic segments in which sequence variation can result in morphological changes and innovation. These elements, conserved over long evolutionary time, emerge as primary candidates that are likely to harbor sequence variation contributing to susceptibility of human disease phenotypes.

Animals↗

PubMed2004

The DNA sequence and comparative analysis of human chromosome 5.

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

Animals↗

PubMed2004

VISTA: computational tools for comparative genomics.

Comparison of DNA sequences from different species is a fundamental method for identifying functional elements in genomes. Here, we describe the VISTA family of tools created to assist biologists in carrying out this task. Our first VISTA server at http://www-gsd.lbl.gov/vista/ was launched in the summer of 2000 and was designed to align long genomic sequences and visualize these alignments with associated functional annotations. Currently the VISTA site includes multiple comparative genomics tools and provides users with rich capabilities to browse pre-computed whole-genome alignments of large vertebrate genomes and other groups of organisms with VISTA Browser, to submit their own sequences of interest to several VISTA servers for various types of comparative analysis and to obtain detailed comparative analysis results for a set of cardiovascular genes. We illustrate capabilities of the VISTA site by the analysis of a 180 kb interval on human chromosome 5 that encodes for the kinesin family member 3A (KIF3A) protein.

Binding Sites↗

PubMed2004

Human apoB contributes to increased serum total apo(a) level in LPA transgenic mice.

BACKGROUND: The Lp(a) lipoprotein (Lp(a)) consists of the polymorphic glycoprotein apolipoprotein(a) (apo(a)), which is attached by a disulfide bond to apolipoprotein B (apoB). Apo(a), which has high homology with plasminogen, is present only in primates and hedgehogs. However, transgenic mice and rabbits with high serum apo(a) levels exist. Liver is the main site for apo(a) synthesis, but the site of removal is uncertain. To examine differences between transgenic mice expressing the LPA gene and mice capable of forming Lp(a) particles, LPA-YAC transgenic mice and hAPOB transgenic mice were crossed and their offspring examined. RESULTS: Comparison of LPA-YAC with LPA-YAC/hAPOB transgenic mice showed that LPA-YAC/hAPOB transgenic mice have higher serum total apo(a) and total cholesterol level than mice lacking the hAPOB gene. However, hepatic apo(a) mRNA level was higher in LPA-YAC transgenic mice than in LPA-YAC/hAPOB transgenic mice. Feeding of a high-cholesterol/high-fat diet to male LPA-YAC transgenic mice with or without the hAPOB gene resulted in reduced serum total apo(a) and hepatic apo(a) mRNA level. CONCLUSION: In conclusion, the higher serum total apo(a) level in LPA-YAC/hAPOB transgenic mice than in LPA-YAC transgenic mice is not caused by increased apo(a) synthesis. Lower hepatic apo(a) mRNA level in LPA-YAC/hAPOB than in LPA-YAC transgenic mice may suggest that the increase in total apo(a) level is a result of apo(a) accumulation in serum. Furthermore, observed higher serum total cholesterol level in LPA-YAC/hAPOB transgenic mice than either in wild type or LPA-YAC transgenic mice may further suggest that human APOB transgenicity is a factor that contributes to increased serum total apo(a) and cholesterol levels. Our results on reduced serum total apo(a) and hepatic apo(a) mRNA levels in HCHF fed male LPA-YAC transgenic mice confirm earlier findings in females, and show that there are no sex difference in mechanisms for lowering apo(a) level in response to HCHF feeding.

Journal Article↗

PubMed2004

Analysis of apolipoprotein A5, c3, and plasma triglyceride concentrations in genetically engineered mice.

OBJECTIVE: Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are strongly altered by changes in the expression of either of these 2 genes. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. These similar findings raised the issue of the relationship between these 2 genes and altered triglycerides. METHODS AND RESULTS: To address this issue, we generated independent lines of mice that either overexpressed ("double transgenic") or completely lacked ("double knockout") both apolipoprotein genes. We report that both "double transgenic" and "double knockout" mice display normal triglyceride concentrations compared with overexpression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the "double transgenic" mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV as a potent triglyceride modulator despite its low concentration. CONCLUSIONS: Together, these data support that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.

Animals↗

PubMed2004

The DNA sequence and biology of human chromosome 19.

Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

Alternative Splicing↗

PubMed2004

Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons.

Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well-established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well-characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL-cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human quantitative trait loci genes in a highly controlled non-human primate model.

Animals↗

PubMed2004

Community structure and metabolism through reconstruction of microbial genomes from the environment.

Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

Archaea↗

PubMed2004

Phylo-VISTA: interactive visualization of multiple DNA sequence alignments.

MOTIVATION: The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. RESULTS: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a framework based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. AVAILABILITY: Phylo-VISTA is available at http://www-gsd.lbl.gov/phylovista. It requires an Internet browser with Java Plug-in 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu

Algorithms↗

PubMed2004

Genetic variation responsible for mouse strain differences in integrin alpha 2 expression is associated with altered platelet responses to collagen.

As mouse models have become commonplace for studying hemostasis and thrombosis, we considered whether the mouse system had utility for assessing genetic alterations in platelet receptors. Platelets from 5 mouse strains (C57BL/6 [C57], FVB/N [FVB], BALB/c, C3H/He, and 129Sv) showed only minor differences in the expression of integrin alpha(IIb), integrin beta(3), glycoprotein (GP) Ib alpha, or GPVI across strains. However, FVB platelets expressed approximately 50% the level of integrin alpha(2) as platelets from other strains (P <.0001). We bred FVB mice with C57 and assessed alpha(2) expression in FVB/C57xFVB/C57 (F2) offspring. Linkage analysis demonstrated the gene responsible for alpha(2) levels is tightly linked to the D13mit260 marker (log odds [lod] score 6.7) near the alpha(2) gene. FVB platelets showed reduced aggregation and a longer lag phase to collagen. FVB and C57 platelets aggregated similarly to collagen-related peptide, but FVB platelets showed a reduction in rhodocytin-induced Syk and PLC gamma 2 tyrosine phosphorylation. Thus, FVB platelets express half the level of alpha(2) as other mouse strains, a trait linked to the alpha(2) gene and seemingly responsible for reduced platelet aggregation to collagen. These strain differences serve as a useful model for the 2-fold difference in human platelet alpha(2)beta(1) expression and demonstrate that alpha(2)beta(1) participates in signaling during platelet activation.

Animals↗

PubMed2004

Identification of a novel enhancer of brain expression near the apoE gene cluster by comparative genomics.

Comparative analysis of the human and mouse genomic sequences downstream of the apolipoprotein E gene (APOE) revealed a highly conserved element with previously undefined function. In reporter gene transfection studies, this element which is located approximately 42 kb distal to APOE was found to have silencer activity in a subset of cell lines examined. Analysis of transgenic mice containing a fusion construct linking this distal 631 bp conserved element to a reporter gene comprised of the human APOE gene with its proximal promoter resulted in robust brain expression of the transgenic human apoE mRNA in three independent transgenic lines, supporting the identification of a novel brain controlling region (BCR). Further studies using immunohistochemistry revealed widespread human apoE localization throughout the brains of the BCR-apoE transgenic mice with prominent expression in the cortex and diencephalon. In addition, double-label immunofluorescence performed on brain sections and cultures of primary cortical cells localized human apoE protein to cortical neurons and microglia. These studies demonstrate that comparative sequence analysis is a successful strategy to predict candidate regulatory regions in vivo, although they do not imply that this element controls apoE expression physiologically.

Animals↗