Biomedical subjects

Lisa Stubbs

Publications and source records attributed to Lisa Stubbs.

At least 19 recordsLinked to original sources

PubMed2006

Identification of clustered YY1 binding sites in imprinting control regions.

Mammalian genomic imprinting is regulated by imprinting control regions (ICRs) that are usually associated with tandem arrays of transcription factor binding sites. In this study, the sequence features derived from a tandem array of YY1 binding sites of Peg3-DMR (differentially methylated region) led us to identify three additional clustered YY1 binding sites, which are also localized within the DMRs of Xist, Tsix, and Nespas. These regions have been shown to play a critical role as ICRs for the regulation of surrounding genes. These ICRs have maintained a tandem array of YY1 binding sites during mammalian evolution. The in vivo binding of YY1 to these regions is allele specific and only to the unmethylated active alleles. Promoter/enhancer assays suggest that a tandem array of YY1 binding sites function as a potential orientation-dependent enhancer. Insulator assays revealed that the enhancer-blocking activity is detected only in the YY1 binding sites of Peg3-DMR but not in the YY1 binding sites of other DMRs. Overall, our identification of three additional clustered YY1 binding sites in imprinted domains suggests a significant role for YY1 in mammalian genomic imprinting.

Alleles↗

PubMed2006

A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors.

Krüppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotes. KRAB-ZNF proteins, in which a potent repressor domain is attached to a tandem array of DNA-binding zinc-finger motifs, are specific to tetrapod vertebrates and represent the largest class of ZNF proteins in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the genome sequence for key motifs and then constructed and manually curated gene models incorporating those sequences. The resulting gene catalog contains 423 KRAB-ZNF protein-coding loci, yielding alternative transcripts that altogether predict at least 742 structurally distinct proteins. Active rounds of segmental duplication, involving single genes or larger regions and including both tandem and distributed duplication events, have driven the expansion of this mammalian gene family. Comparisons between the human genes and ZNF loci mined from the draft mouse, dog, and chimpanzee genomes not only identified 103 KRAB-ZNF genes that are conserved in mammals but also highlighted a substantial level of lineage-specific change; at least 136 KRAB-ZNF coding genes are primate specific, including many recent duplicates. KRAB-ZNF genes are widely expressed and clustered genes are typically not coregulated, indicating that paralogs have evolved to fill roles in many different biological processes. To facilitate further study, we have developed a Web-based public resource with access to gene models, sequences, and other data, including visualization tools to provide genomic context and interaction with other public data sets.

Computational Biology↗

PubMed2006

Evolutionary expansion and divergence in the ZNF91 subfamily of primate-specific zinc finger genes.

Most genes are conserved in mammals, but certain gene families have acquired large numbers of lineage-specific loci through repeated rounds of gene duplication, divergence, and loss that have continued in each mammalian group. One such family encodes KRAB-zinc finger (KRAB-ZNF) proteins, which function as transcriptional repressors. One particular subfamily of KRAB-ZNF genes, including ZNF91, has expanded specifically in primates to comprise more than 110 loci in the human genome. Genes of the ZNF91 subfamily reside in large gene clusters near centromeric regions of human chromosomes 19 and 7 with smaller clusters or isolated copies in other locations. Phylogenetic analysis indicates that many of these genes arose before the split between the New and Old World monkeys, but the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. Paralogous loci are distinguished by divergence within their zinc finger arrays, indicating selection for proteins with different regulatory targets. In addition, many loci produce multiple alternatively spliced transcripts encoding proteins that may serve separate and perhaps even opposing regulatory roles because of the modular motif structure of KRAB-ZNF genes. The tissue-specific expression patterns and rapid structural divergence of ZNF91 subfamily genes suggest a role in determining gene expression differences between species and the evolution of novel primate traits.

Animals↗

PubMed2006

Allele-specific deposition of macroH2A1 in imprinting control regions.

In the current study, we analyzed the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes. MacroH2A1 is preferentially deposited at methylated CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the imprinting control regions (ICRs) of Xist, Peg3, H19/Igf2, Gtl2/Dlk1 and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other differentially methylated regions of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. Overall, our results indicate that besides DNA methylation macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles.

Alleles↗

PubMed2005

Rapid evolution of a recently retroposed transcription factor YY2 in mammalian genomes.

YY2 was originally identified due to its unusual similarity to the evolutionarily well-conserved zinc finger gene YY1. In this study, we have determined the evolutionary origin and conservation of YY2 using comparative genomic approaches. Our results indicate that YY2 is a retroposed copy of YY1 that has been inserted into another gene locus named Mbtps2 (membrane-bound transcription factor protease site 2). This retroposition is estimated to have occurred after the divergence of placental mammals from other vertebrates based on the detection of YY2 only in the placental mammals. The N- and C-terminal regions of YY2 have evolved under different selection pressures. The N-terminal region has evolved at a very fast pace with very limited functional constraints, whereas the DNA-binding, C-terminal region still maintains a sequence structure very similar to that of YY1 and is also well conserved among placental mammals. In situ hybridizations using different adult mouse tissues indicate that mouse YY2 is expressed at relatively low levels in Purkinje and granular cells of cerebellum and in neuronal cells of cerebrum, but at very high levels in testis. The expression levels of YY2 are much lower than those of YY1, but the overall spatial expression patterns are similar to those of Mbtps2, suggesting a possible shared transcriptional control between YY2 and Mbtps2. Taken together, the formation and evolution of YY2 represent a very unusual case where a transcription factor was first retroposed into another gene locus encoding a protease and survived with different selection schemes and expression patterns.

Amino Acid Sequence↗

PubMed2005

A mutation in NFkB interacting protein 1 results in cardiomyopathy and abnormal skin development in wa3 mice.

We have identified waved 3 (wa3), a novel recessive mutation that causes abnormalities of the heart and skin. The cardiac defect results in a severe and rapidly progressive dilated cardiomyopathy. We identified the gene mutated in these mice, which we call NFkB interacting protein1 (Nkip1), using positional cloning. Nkip1 is expressed in skin, heart and vascular endothelium and shares homology with a small family of proteins that play a role in the regulation of transcription factors. A C-terminal fragment of this protein was previously identified as the RelA associated inhibitor (RAI). We show that the full-length protein is larger than previously described, and we confirm that it interacts with NFkB in vivo. Expression analysis of genes known to be regulated by NFkB revealed that Intercellular adhesion molecule 1 (Icam1) expression is consistently elevated in mutant mice. This result suggests that wa3 mutant mice represent a potentially important model for the analysis of the role of inflammatory processes in heart disease.

Animals↗

PubMed2005

Two reciprocal translocations provide new clues to the high mutability of the Grid2 locus.

We describe two new mutations, 153Gso and 154Gso, associated with reciprocal translocations with a common breakpoint in mouse chromosome 6B3 (Mmu6B3). The translocations arose independently in offspring of male mice treated with chlorambucil and glycidamide, respectively. Homozygotes of both mutant stocks display a characteristic gait ataxia with 'foot-patting' behavior; despite their ataxia the mutant animals are healthy, long-lived, and breed normally. Breeding experiments confirmed that 153Gso and 154Gso mutations are allelic, and both fail to complement a known mutation hotfoot (ho), a Mmu6 mutation involving the glutamate receptor gene, Grid2, that is associated with a virtually identical phenotype. Our studies demonstrate that the 153Gso and 154Gso mutations disrupt the Grid2 gene at sites located more than 100 kb apart in intron 6 and intron 4 of the gene, respectively. The occurrence of two independent translocations from a relatively small colony within the same locus supports data suggesting the hypermutability of the Grid2 locus and suggest that the gene's large size make it an especially likely target for mutations involving genetic rearrangement.

Animals↗

PubMed2004

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms.

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms (SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds (a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines--in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases.

Alleles↗

PubMed2004

Mulan: multiple-sequence local alignment and visualization for studying function and evolution.

Multiple-sequence alignment analysis is a powerful approach for understanding phylogenetic relationships, annotating genes, and detecting functional regulatory elements. With a growing number of partly or fully sequenced vertebrate genomes, effective tools for performing multiple comparisons are required to accurately and efficiently assist biological discoveries. Here we introduce Mulan (http://mulan.dcode.org/), a novel method and a network server for comparing multiple draft and finished-quality sequences to identify functional elements conserved over evolutionary time. Mulan brings together several novel algorithms: the TBA multi-aligner program for rapid identification of local sequence conservation, and the multiTF program for detecting evolutionarily conserved transcription factor binding sites in multiple alignments. In addition, Mulan supports two-way communication with the GALA database; alignments of multiple species dynamically generated in GALA can be viewed in Mulan, and conserved transcription factor binding sites identified with Mulan/multiTF can be integrated and overlaid with extensive genome annotation data using GALA. Local multiple alignments computed by Mulan ensure reliable representation of short- and large-scale genomic rearrangements in distant organisms. Mulan allows for interactive modification of critical conservation parameters to differentially predict conserved regions in comparisons of both closely and distantly related species. We illustrate the uses and applications of the Mulan tool through multispecies comparisons of the GATA3 gene locus and the identification of elements that are conserved in a different way in avians than in other genomes, allowing speculation on the evolution of birds. Source code for the aligners and the aligner-evaluation software can be freely downloaded from http://www.bx.psu.edu/miller_lab/.

Animals↗

PubMed2004

Evolution and functional classification of vertebrate gene deserts.

Large tracts of the human genome, known as gene deserts, are devoid of protein-coding genes. Dichotomy in their level of conservation with chicken separates these regions into two distinct categories, stable and variable. The separation is not caused by differences in rates of neutral evolution but instead appears to be related to different biological functions of stable and variable gene deserts in the human genome. Gene Ontology categories of the adjacent genes are strongly biased toward transcriptional regulation and development for the stable gene deserts, and toward distinctively different functions for the variable gene deserts. Stable gene deserts resist chromosomal rearrangements and appear to harbor multiple distant regulatory elements physically linked to their neighboring genes, with the linearity of conservation invariant throughout vertebrate evolution.

Animals↗

PubMed2004

ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes.

With an increasing number of vertebrate genomes being sequenced in draft or finished form, unique opportunities for decoding the language of DNA sequence through comparative genome alignments have arisen. However, novel tools and strategies are required to accommodate this large volume of genomic information and to facilitate the transfer of predictions generated by comparative sequence alignment to researchers focused on experimental annotation of genome function. Here, we present the ECR Browser, a tool that provides easy and dynamic access to whole genome alignments of human, mouse, rat and fish sequences. This web-based tool (http://ecrbrowser.dcode.org) provides the starting point for discovery of novel genes, identification of distant gene regulatory elements and prediction of transcription factor binding sites. The genome alignment portal of the ECR Browser also permits fast and automated alignments of any user-submitted sequence to the genome of choice. The interconnection of the ECR Browser with other DNA sequence analysis tools creates a unique portal for studying and exploring vertebrate genomes.

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

Lineage-specific imprinting and evolution of the zinc-finger gene ZIM2.

We have carried out an in-depth comparative analysis of a 100-kb genomic interval containing two imprinted genes, PEG3 and ZIM2, using sequences derived from human, mouse, and cow. In all three mammals, ZIM2 is located at a similar genomic distance and in the same orientation relative to PEG3, indicating the basic structural conservation of this imprinted locus. However, several lineage-specific changes have occurred that affect the exon structure and imprinting status of ZIM2. Human ZIM2 and PEG3 share a set of 5' exons and a common promoter, and both genes are paternally expressed. In contrast, mouse and cow Zim2 genes do not share 5' exons with Peg3, and Zim2 employs a separate downstream promoter in both species. The imprinting status of Zim2 is also not conserved among mammals; mouse Zim2 is expressed biallelically in testis but predominantly from the maternal allele in brain, while cow Zim2 is expressed biallelically in testis. The separate transcription of Zim2 and Peg3 and the change in promoter usage and imprinting status appear to have resulted from independent insertional events that have placed unrelated genes, Zim1 and Ast1, respectively, between Zim2 and Peg3 in mouse and cow. Our results suggest that PEG3 and ZIM2 represent the two original genes at this locus and that rearrangements have occurred independently in different mammalian lineages in recent evolutionary times. Our data also suggest that exon-sharing of human PEG3 and ZIM2 was not ancestral, but may represent a fusion event joining the two neighboring genes and bringing ZIM2 under paternal expression control. These observations are striking in light of the structural and functional conservation that typifies other imprinted domains and suggest that the PEG3/ZIM2 imprinted domain may have evolved in an unusual lineage-specific pattern.

Amino Acid Sequence↗

PubMed2004

Interpreting mammalian evolution using Fugu genome comparisons.

Recently, it has been shown that a significant number of evolutionarily conserved human-Fugu noncoding elements function as tissue-specific transcriptional enhancers in vivo, suggesting that distant comparisons are capable of identifying a particular class of regulatory elements. We therefore hypothesized that by juxtaposing human/Fugu and human/mouse conservation patterns we can define conservation criteria for discovering transcriptional regulatory elements specific to mammals. Genome-scale comparisons of noncoding human/Fugu evolutionary conserved elements (ECRs) and their humans/mouse counterparts revealed a particular signature common to human/mouse ECRs (>or=350 bp long, >or=77% identity) that are also conserved in fishes. This newly defined threshold identifies 90% of all human/Fugu noncoding ECRs without the assistance of human-Fugu genome alignments and provides a very efficient filter for identifying functional human/mouse ECRs.

Animals↗

PubMed2004

zPicture: dynamic alignment and visualization tool for analyzing conservation profiles.

Comparative sequence analysis has evolved as an essential technique for identifying functional coding and noncoding elements conserved throughout evolution. Here, we introduce zPicture, an interactive Web-based sequence alignment and visualization tool for dynamically generating conservation profiles and identifying evolutionarily conserved regions (ECRs). zPicture is highly flexible, because critical parameters can be modified interactively, allowing users to differentially predict ECRs in comparisons of sequences of different phylogenetic distances and evolutionary rates. We demonstrate the application of this module to identify a known regulatory element in the HOXD locus, in which functional ECRs are difficult to discern against the highly conserved genomic background. zPicture also facilitates transcription factor binding-site analysis via the rVista tool portal. We present an example of the HBB complex when zPicture/rVista combination specifically pinpoints to two ECRs containing GATA-1, NF-E2, and TAL1/E47 binding sites that were identified previously as transcriptional enhancers. In addition, zPicture is linked to the UCSC Genome Browser, allowing users to automatically extract sequences and gene annotations for any recorded locus. Finally, we describe how this tool can be efficiently applied to the analysis of nonvertebrate genomes, including those of microbial organisms.

Animals↗

PubMed2003

Differential expansion of zinc-finger transcription factor loci in homologous human and mouse gene clusters.

Mammalian genomes carry hundreds of Krüppel-type zinc finger (ZNF) genes, most of which reside in familial clusters. ZNF genes encoding Krüppel-associated box (KRAB) motifs are especially prone to this type of tandem organization. Despite their prevalence, little is known about the functions or evolutionary histories of these clustered gene families. Here we describe a homologous pair of human and mouse KRAB-ZNF gene clusters containing 21 human and 10 mouse genes, respectively. Evolutionary analysis uncovered only three pairs of putative orthologs and two cases where a single gene in one species is related to multiple genes in the other; several human genes have no obvious homolog in mouse. We deduce that duplication and loss of ancestral cluster members occurred independently in the primate and rodent lineages after divergence, yielding substantially different ZNF gene repertoires in humans and mice. Differences in expression patterns and sequence divergence within the DNA binding regions of predicted proteins suggest that the duplicated genes have acquired novel functions over evolutionary time. Since KRAB-ZNF proteins are predicted to function as transcriptional regulators, the elaboration of new lineage-specific genes in this and other clustered ZNF families is likely to have had a significant impact on species-specific aspects of biology.

Amino Acid Sequence↗

PubMed2003

Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3.

The 5'-ends of two paternally expressed mouse genes, Peg3 and Usp29, are jointly associated with a CpG island that exhibits allele-specific methylation. Sequence comparison of the regions derived from human, mouse and cow revealed the presence of two evolutionarily conserved sequence motifs including one that is repeated multiple times within the first intron of Peg3 in all three mammals. DNA mobility shift and chromatin immunoprecipitation (ChIP) assays clearly demonstrated that this motif is an in vivo binding site for the Gli-type transcription factor YY1. The YY1-binding site contains one CpG dinucleotide, and methylation of this CpG site abolishes the binding activity of YY1 in vitro. The Peg3 YY1-binding sites are methylated only on the maternal chromosome in vivo, and ChIP assays confirmed that YY1 binds specifically to the paternal allele of the gene. Promoter, enhancer and insulator assays with deletion constructs of sequence surrounding the YY1-binding sites indicate that the region functions as a methylation-sensitive insulator that may influence the imprinted expression of Peg3 and neighboring genes. The current study is the first report demonstrating the involvement of YY1 in methylation-sensitive insulator activity and suggests a potential role of this highly conserved protein in mammalian genomic imprinting.

Animals↗

PubMed2002

EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages, binds to a putative cellular ligand on B lymphoma cell line A20.

A novel member of the EGF-TM7 family, mEMR4, was identified and characterized. The full-length mouse EMR4 cDNA encodes a predicted 689-amino acid protein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localized in the distal region of mouse chromosome 17 in close proximity to another EGF-TM7 gene, F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in Biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-alpha-treated resident peritoneal macrophages, whereas interleukin-4 and -10 dramatically reduce the expression. mEMR4 was found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated noncovalently as a heterodimer. The proteolytic cleavage site was identified by N-terminal amino acid sequencing and located at the conserved GPCR (G protein-coupled receptor) proteolytic site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.

Amino Acid Sequence↗