Biomedical subjects

Shawn M Burgess

Publications and source records attributed to Shawn M Burgess.

12 recordsLinked to original sources

PubMed2006

The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15.

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation.

Animals↗

PubMed2006

The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure.

All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites.

Animals↗

PubMed2005

Redefining the common insertion site.

Retroviral mutagenesis has been used as a powerful tool to discover genes involved in oncogenesis through a technique called Common Insertion Site (CIS) analysis where tumors are induced by proviral integrations and the genomic loci of the proviruses are identified. A fundamental assumption made in this analysis is that multiple proviral insertions in close proximity occurring more frequently than would be predicted randomly provides evidence that the genes near the integrations are involved in the formation of the tumors. We demonstrate here using data derived from MLV integrations not put under selection for tumor induction that CIS analysis as currently defined is often not a sufficient argument for a gene's significance in tumorigenesis.

Gene Expression Regulation↗

PubMed2005

The zebrafish gene claudinj is essential for normal ear function and important for the formation of the otoliths.

We have identified a mutation in the zebrafish gene claudinj generated by retroviral integration. Mutant embryos display otoliths severely reduced in size, no response to tapping stimulus, and an inability to balance properly suggesting vestibular and hearing dysfunction. Antisense in situ hybridization to the cldnj gene showed expression first in the otic placode and later asymmetric expression in the otic vesicle. Morpholino inhibition of claudinj expression showed similar defects in otolith formation. Phylogenetic analysis of claudin sequences from multiple species demonstrates that claudinj was part of a gene expansion that began in the common ancestor of fish and humans, but additional fish specific gene duplications must have also occurred.

Animals↗

PubMed2005

Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.

Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

Animals↗

PubMed2005

Weak palindromic consensus sequences are a common feature found at the integration target sites of many retroviruses.

Integration into the host genome is one of the hallmarks of the retroviral life cycle and is catalyzed by virus-encoded integrases. While integrase has strict sequence requirements for the viral DNA ends, target site sequences have been shown to be very diverse. We carefully examined a large number of integration target site sequences from several retroviruses, including human immunodeficiency virus type 1, simian immunodeficiency virus, murine leukemia virus, and avian sarcoma-leukosis virus, and found that a statistical palindromic consensus, centered on the virus-specific duplicated target site sequence, was a common feature at integration target sites for these retroviruses.

Base Sequence↗

PubMed2005

High-resolution genome-wide mapping of transposon integration in mammals.

The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon- and virus-based integrating vectors.

Animals↗

PubMed2004

Oculofaciocardiodental and Lenz microphthalmia syndromes result from distinct classes of mutations in BCOR.

Lenz microphthalmia is inherited in an X-linked recessive pattern and comprises microphthalmia, mental retardation, and skeletal and other anomalies. Two loci associated with this syndrome, MAA (microphthalmia with associated anomalies) and MAA2, are situated respectively at Xq27-q28 (refs. 1,2) and Xp11.4-p21.2 (ref. 3). We identified a substitution, nt 254C-->T; P85L, in BCOR (encoding BCL-6-interacting corepressor, BCOR) in affected males from the family with Lenz syndrome previously used to identify the MAA2 locus. Oculofaciocardiodental syndrome (OFCD; OMIM 300166) is inherited in an X-linked dominant pattern with presumed male lethality and comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. Given their phenotypic overlap, we proposed that OFCD and MAA2-associated Lenz microphthalmia were allelic, and we found different frameshift, deletion and nonsense mutations in BCOR in seven families affected with OFCD. Like wild-type BCOR, BCOR P85L and an OFCD-mutant form of BCOR can interact with BCL-6 and efficiently repress transcription. This indicates that these syndromes are likely to result from defects in alternative functions of BCOR, such as interactions with transcriptional partners other than BCL-6. We cloned the zebrafish (Danio rerio) ortholog of BCOR and found that knock-down of this ortholog caused developmental perturbations of the eye, skeleton and central nervous system consistent with the human syndromes, confirming that BCOR is a key transcriptional regulator during early embryogenesis.

Abnormalities, Multiple↗

PubMed2004

Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids.

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.

Cell Division↗

PubMed2004

Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm.

Transgenic modification of sperm before fertilization has distinct advantages over conventional transgenic methods. The primary advantage is that the mosaicism inherent in those other techniques is avoided. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation from spermatogonia to functional sperm can occur in vitro, provides the opportunity for genetic modification of sperm in vitro. Here, we report the production of transgenic zebrafish from cultured sperm. The sperm were differentiated from premeiotic germ cells infected with a pseudotyped retrovirus in vitro. The collected sperm were used to perform successful in vitro fertilizations, and transgenic embryos were identified. The transgenic fish transmitted the proviral integration to the next generation in a Mendelian fashion. We report the generation of a transgenic animal by cultured sperm and open the door to many exciting possibilities for the rapid generation of transgenic lines in model organisms such as zebrafish or other animal systems that are otherwise intractable to transgenesis.

Animals↗

PubMed2003

Transcription start regions in the human genome are favored targets for MLV integration.

Factors contributing to retroviral integration have been intractable because past studies have not precisely located genomic sites of proviruses in sufficient numbers for significant analysis. In this study, 903 murine leukemia virus (MLV) and 379 human immunodeficiency virus-1 (HIV-1) integrations in the human genome were mapped. The data showed that MLV preferred integration near the start of transcriptional units (either upstream or downstream) whereas HIV-1 preferred integration anywhere in the transcriptional unit but not upstream of the transcriptional start. Defining different integration site preferences for retroviruses will have important ramifications for gene therapy and may aid in our understanding of the factors directing the integration process.

Child↗

PubMed2003

Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning.

We identified four insertional alleles of foxi one (foo), an embryonic lethal mutation in zebrafish that displays defects in both otic placode and the jaw. In foo/foo embryos the otic placode is split into two smaller placodes and mutant embryos show a dorsoventral (DV) cartilage defect manifested as a reduced hyomandibular and reduced third and fourth branchial arches. We identified foxi one (foo), the zebrafish ortholog of Foxi1 (FREAC6, FKHL10, HFH-3, Fkh10) and a member of the forkhead domain transcriptional regulator family, as the gene mutated in foo/foo embryos. foo is expressed in otic placode precursor cells, and foo/foo embryos lack placodal pax8 expression and have disorganized otic expression of pax2.1 and dlx3. Third stream neural crest cell migration, detected by dlx2 and krox20 expression, is aberrant in that it invades the otic placode territory. foo is expressed in pharyngeal pouch endoderm and is required for pouch expression of pax8 and proper patterning of other markers in the pouch such as nkx2.3. In foo/foo embryos, we observed a failure to maintain fgf3 expression in the pouches, followed by apoptosis of neural crest cells in adjacent arches. We conclude that foo expression is essential for pax8 expression probably downstream of Fgf signaling in a conserved pathway jointly required for integrity of patterning in the otic placode and pharyngeal pouches. We propose that correct placement of survival/proliferation cues is essential for shaping the pharyngeal cartilages and that evolutionary links between jaw and ear formation can be traced to Fgf-Foxi1-Pax8 pathways.

Alleles↗