Biomedical subjects

Michael O Dorschner

Publications and source records attributed to Michael O Dorschner.

8 recordsLinked to original sources

PubMed2006

Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays.

Localized accessibility of critical DNA sequences to the regulatory machinery is a key requirement for regulation of human genes. Here we describe a high-resolution, genome-scale approach for quantifying chromatin accessibility by measuring DNase I sensitivity as a continuous function of genome position using tiling DNA microarrays (DNase-array). We demonstrate this approach across 1% ( approximately 30 Mb) of the human genome, wherein we localized 2,690 classical DNase I hypersensitive sites with high sensitivity and specificity, and also mapped larger-scale patterns of chromatin architecture. DNase I hypersensitive sites exhibit marked aggregation around transcriptional start sites (TSSs), though the majority mark nonpromoter functional elements. We also developed a computational approach for visualizing higher-order features of chromatin structure. This revealed that human chromatin organization is dominated by large (100-500 kb) 'superclusters' of DNase I hypersensitive sites, which encompass both gene-rich and gene-poor regions. DNase-array is a powerful and straightforward approach for systematic exposition of the cis-regulatory architecture of complex genomes.

Chromatin↗

PubMed2004

High-throughput localization of functional elements by quantitative chromatin profiling.

Identification of functional, noncoding elements that regulate transcription in the context of complex genomes is a major goal of modern biology. Localization of functionality to specific sequences is a requirement for genetic and computational studies. Here, we describe a generic approach, quantitative chromatin profiling, that uses quantitative analysis of in vivo chromatin structure over entire gene loci to rapidly and precisely localize cis-regulatory sequences and other functional modalities encoded by DNase I hypersensitive sites. To demonstrate the accuracy of this approach, we analyzed approximately 300 kilobases of human genome sequence from diverse gene loci and cleanly delineated functional elements corresponding to a spectrum of classical cis-regulatory activities including enhancers, promoters, locus control regions and insulators as well as novel elements. Systematic, high-throughput identification of functional elements coinciding with DNase I hypersensitive sites will substantially expand our knowledge of transcriptional regulation and should simplify the search for noncoding genetic variation with phenotypic consequences.

Algorithms↗

PubMed2004

Discovery of functional noncoding elements by digital analysis of chromatin structure.

We developed a quantitative methodology, digital analysis of chromatin structure (DACS), for high-throughput, automated mapping of DNase I-hypersensitive sites and associated cis-regulatory sequences in the human and other complex genomes. We used 19/20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced approximately 257,000 tags from erythroid cells. Tags were mapped to the human genome, and a quantitative algorithm was applied to discriminate statistically significant clusters of independent DNase I cutting events. We show that such clusters identify both known regulatory sequences and previously unrecognized functional elements across the genome. We used in silico simulation to demonstrate that DACS is capable of efficient and accurate localization of the majority of DNase I-hypersensitive sites in the human genome without requiring an independent validation step. A unique feature of DACS is that it permits unbiased evaluation of the chromatin state of regulatory sequences from widely separated genomic loci. We found surprisingly large differences in the accessibility of distant regulatory sequences, suggesting the existence of a hierarchy of nuclear organization that escapes detection by conventional chromatin assays.

Chromatin↗

PubMed2004

Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries.

Comprehensive identification of sequences that regulate transcription is one of the major goals of genome biology. Focal alteration in chromatin structure in vivo, detectable through hypersensitivity to DNaseI and other nucleases, is the sine qua non of a diverse cast of transcriptional regulatory elements including enhancers, promoters, insulators, and locus control regions. We developed an approach for genome-scale identification of DNaseI hypersensitive sites (HSs) via isolation and cloning of in vivo DNaseI cleavage sites to create libraries of active chromatin sequences (ACSs). Here, we describe analysis of >61,000 ACSs derived from erythroid cells. We observed peaks in the density of ACSs at the transcriptional start sites of known genes at non-gene-associated CpG islands, and, to a lesser degree, at evolutionarily conserved noncoding sequences. Peaks in ACS density paralleled the distribution of DNaseI HSs. ACSs and DNaseI HSs were distributed between both expressed and nonexpressed genes, suggesting that a large proportion of genes reside within open chromatin domains. The results permit a quantitative approximation of the distribution of HSs and classical cis-regulatory sequences in the human genome.

Chromatin↗

PubMed2004

Genomic context of paralogous recombination hotspots mediating recurrent NF1 region microdeletion.

Recombination between paralogs that flank the NF1 gene at 17q11.2 typically results in a 1.5-Mb microdeletion that includes NF1 and at least 13 other genes. We show that the principal sequences responsible are two 51-kb blocks with 97.5% sequence identity (NF1REP-P1-51 and NF1REP-M-51). These blocks belong to a complex group of paralogs with three components on 17q11.2 and another on 19p13.13. Breakpoint sequencing of deleted chromosomes from multiple patients revealed two paralogous recombination hot spots within the 51-kb blocks. Lack of sequence similarity between these sites failed to suggest or corroborate any putative cis-acting recombinogenic motifs. However, the NF1 REPs showed relatively high alignment mismatch between recombining paralogs, and we note that the NF1REP hot spots were regions of good alignment bordered by relatively large alignment gaps. Statistical tests for gene conversion detected a single significant tract of perfect match between the NF1REPs that was 700 bp long and coincided with PRS2, the predominant recombination hot spot. Tracts of perfect match occurring by chance may contribute to breakpoint localization, but our result suggests that perfect tracts at recombination hot spots may be a result of gene conversion at sites at which preferential pairing occurs for other, as-yet-unknown reasons.

Base Sequence↗

PubMed2003

Complete physical map and gene content of the human NF1 tumor suppressor region in human and mouse.

Duplicon-mediated microdeletions around the NF1 gene are frequently associated with a severe form of neurofibromatosis type I in a subgroup of patients who show an earlier onset of cutaneous neurofibromas, dysmorphic facial features, and lower IQ values. To clarify the discrepancies between published maps of the NF1 tumor-suppressor gene region as well as the length of gaps in these assemblies and to validate the recently described tandem duplication of the human NF1 locus, we assembled a contiguous high-density map of BAC and PAC clones from different genomic libraries. Although two WI-12393-derived low-copy fragments are known to occur at the proximal and distal boundaries of the 1.5-Mb segment that is usually deleted in NF1 microdeletion patients, we identified an additional WI-12393-related segment between the MGC13061 and the NF1 gene, which appears to trigger interstitial deletions of smaller size as observed in two patients. Moreover, we completed the genomic organization and cDNA structure of all functional genes, CYTOR4, FLJ12735, FLJ22729, CENTA2, MGC13061, NF1, OMG, EVI2B, EVI2A, KIAA1821, MGC11316, HCA66, KIAA0160, and WI-12393, from this region. A comparison of the human map to the orthologous region on mouse chromosome 11 revealed significant differences in the number and arrangement of genes, indicating that many chromosomal breaks with partial duplications, inversions, and deletions occurred predominantly in the primate lineage.

Adult↗

PubMed2002

The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1).

Iron is an essential nutrient required for the function of all cells, most notably for the production of hemoglobin in red blood cells. Defects in the mechanisms of iron absorption, storage, or utilization can lead to disorders of iron-limited erythropoiesis or iron overload. In an effort to further understand these processes, we have used the zebrafish as a genetic system to study vertebrate iron metabolism. Here we characterized the phenotype of chardonnay (cdy), a zebrafish mutant with hypochromic, microcytic anemia, and positioned the mutant gene on linkage group 11. The cdy gene was isolated by a functional genomics approach in which we used a combination of expression studies, sequence analyses, and radiation hybrid panel mapping. We identified erythroid-specific genes using a whole embryo mRNA in situ hybridization screen and placed these genes on the zebrafish genomic map. One of these genes encoded the iron transporter divalent metal transporter 1 (DMT1) and colocalized with the cdy gene. We identified a nonsense mutation in the cdy allele and demonstrated that, whereas wild-type zebrafish DMT1 protein can transport iron, the truncated protein expressed in cdy mutants is not functional. Our studies further demonstrate the conservation of iron metabolism in vertebrates and suggest the existence of an alternative pathway of intestinal and red blood cell iron uptake.

Alleles↗

PubMed2002

Diagnosis of five spinocerebellar ataxia disorders by multiplex amplification and capillary electrophoresis.

The autosomal-dominant spinocerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders with variable expression and phenotypic overlap. An accurate diagnosis relies on detection of a mutation in a specific causative gene, which is typically an abnormal number of CAG trinucleotide repeats. To streamline testing in a clinical setting, we converted our current panel of tests for the spinocerebellar ataxias (SCA) types SCA1, SCA2, SCA3, SCA6, and SCA7 from five independent amplification reactions analyzed by polyacrylamide gel electrophoresis (PAGE) to a single multiplex amplification reaction analyzed by capillary electrophoresis (CE). Multiplex amplification was facilitated by the use of chimeric primers; different lengths and fluorochromes distinguished the amplicons. During CE with commercially available molecular weight standards, the SCA amplicons migrated faster than predicted, thereby underestimating their length compared to that determined previously by PAGE. This was observed to varying degrees for each of the five loci, with the greatest size differential occurring in amplicons with greater (CAG)(n). To determine accurate amplicon length, and therefore an accurate number of CAG repeats, a size correction formula was calculated for each locus. This multiplex semi-automated assay has been reliable during 1 year of use in a clinical setting during which 57 samples were tested and five positive samples were detected.

Ataxin-1↗