Roche NimbleGen Webinar Series

A second project explores the function of PTEN and other components of this important growth regulatory pathway in neural development, using Drosophila as our model system. We document that the PTEN-TSC-TOR signaling pathway is critical for both axon guidance and synapse assembly. Hyperactivation of this pathway can be genetically or pharmacologically separated from growth effects, yet still produce disruption of neural development, indicating this signaling system has growth-independent roles in neural development. We have also documented behavioral deficits in adult flies with modest activation of this pathway in the nervous system, suggesting Drosophila could provide a useful model for tuberous sclerosis complex.

A second project explores the function of PTEN and other components of this important growth regulatory pathway in neural development, using Drosophila as our model system. We document that the PTEN-TSC-TOR signaling pathway is critical for both axon guidance and synapse assembly. Hyperactivation of this pathway can be genetically or pharmacologically separated from growth effects, yet still produce disruption of neural development, indicating this signaling system has growth-independent roles in neural development. We have also documented behavioral deficits in adult flies with modest activation of this pathway in the nervous system, suggesting Drosophila could provide a useful model for tuberous sclerosis complex.

We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50Kb) multigene deletion and a small (1Kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8Kb deletion targeting the ast-1 gene on chromosome II and another is a 141bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to 750Kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira.

We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50Kb) multigene deletion and a small (1Kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8Kb deletion targeting the ast-1 gene on chromosome II and another is a 141bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to >750Kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira.

Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome.

Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome.

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.

Audio: Genome-Wide DNA Methylation Analysis with Immunoprecipitation and Tiling DNA Microarrays

Video: Genome-Wide DNA Methylation Analysis with Immunoprecipitation and Tiling DNA Microarrays

Audio: NimbleGen ASHG 2006 Workshop - Full Program

Video: NimbleGen ASHG 2006 Workshop - Full Program

Audio: ASHG 2006 Presentation: The Discovery and Characterization of Genomic Disorders with High-Resolution Array CGH

Video: ASHG 2006 Presentation: The Discovery and Characterization of Genomic Disorders with High-Resolution Array CGH

Audio: Cytosine Methylation Dynamics in Development and Disease