Minisatellites Show Rare and Simple Intra-allelic Instability in the Mouse Germ Line

Genomics. Jul, 2002  |  Pubmed ID: 12079275

Minisatellites provide very informative systems for analyzing processes of tandem repeat DNA turnover in humans. The mouse genome also contains authentic minisatellites, but none has yet been found to show high levels of instability. Indirect evidence using minisatellite variant repeat mapping by PCR in Mus musculus subspecies suggested that mouse minisatellites mutate at a rate below 10(-3) per gamete and mainly by intra-allelic events. This is in sharp contrast to the complex interallelic mutations observed at high frequency at some human loci. To define more directly the turnover mechanisms and rates of instability at one of the most variable mouse minisatellites (MMS80), we used size-enrichment small-pool PCR (SESP-PCR) to recover de novo mutant alleles from sperm DNA from homozygous BALB/cJ mice and from strain DHA heterozygotes. The sperm mutation rate at MMS80 was extremely low, at or below 5 x 10(-6) per sperm. Comparison of progenitor and mutant allele structures showed that these rare mutants had arisen by simple and primarily, if not exclusively, intra-allelic mutation events. These results suggest a fundamental difference in turnover mechanisms at minisatellites between mice and human.

FKHR (FOXO1a) is Required for Myotube Fusion of Primary Mouse Myoblasts

The EMBO Journal. Mar, 2003  |  Pubmed ID: 12606579

Activation of the transcription factor FKHR (Forkhead in human rhabdomyosarcoma, FOXO1a) in various established cell lines induces cell cycle arrest followed by apoptosis. These effects are inhibited through activation of the phosphatidylinositol 3-kinase/Akt pathway, resulting in FKHR phosphorylation and its export from the nucleus, thus blocking its pro-apoptotic activity. Here we report that FKHR regulates fusion of differentiating primary myoblasts. We demonstrate that FKHR is localized in the cytoplasm of proliferating myoblasts, yet translocates to the nucleus by a phosphorylation-independent pathway following serum starvation, a condition that induces myoblast differentiation. FKHR phosphorylation during terminal differentiation appears to downregulate its fusion activity, as a dominant-active non-phosphorylatable FKHR mutant dramatically augments the rate and extent of myotube fusion. However, this FKHR mutant exerts its effects only after other events initiated the differentiation pro cess. Conversely, enforced expression of a dominant-negative FKHR mutant blocks myotube formation whereas wild-type FKHR has no effect. We conclude that in addition to the role of FoxO proteins in regulating cell cycle progress and apoptosis, FKHR controls the rate of myotube fusion during myogenic differentiation.

Hypermutable Minisatellites, a Human Affair?

Genomics. Apr, 2003  |  Pubmed ID: 12676558

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.

Vinculin Activation by Talin Through Helical Bundle Conversion

Nature. Jan, 2004  |  Pubmed ID: 14702644

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.

Crystal Structure of Human Vinculin

Structure (London, England : 1993). Jul, 2004  |  Pubmed ID: 15242595

Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large number of cytoskeletal and signaling proteins, and this flexibility is thought to contribute to rapid dissociation and reassociations of adhesion complexes. Intramolecular interactions between vinculin's head (Vh) and tail (Vt) domains limit access of its binding sites for other adhesion proteins. While the crystal structures of the Vh and Vt domains are known, these domains represent less than half of the entire protein and are separated by a large central region of unknown structure and function. Here we report the crystal structure of human full-length vinculin to 2.85 A resolution. In its resting state, vinculin is a loosely packed collection of alpha-helical bundles held together by Vh-Vt interactions. The three new well ordered alpha-helical bundle domains are similar in their structure to either Vh (Vh2 and Vh3) or to Vt (Vt2) and their loose packing provides the necessary flexibility that allows vinculin to interact with its various protein partners at sites of cell adhesion.

Structural Dynamics of Alpha-actinin-vinculin Interactions

Molecular and Cellular Biology. Jul, 2005  |  Pubmed ID: 15988023

Alpha-actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. alpha-Actinin interacts with vinculin through the binding of an alpha-helix (alphaVBS) present within the R4 spectrin repeat of its central rod domain to vinculin's N-terminal seven-helical bundle domain (Vh1). The Vh1:alphaVBS structure suggests that alphaVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. alphaVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculin's tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, alphaVBS binds to Vh1 in an inverted orientation compared to the binding of talin's VBSs to vinculin. Importantly, the binding of alphaVBS and talin's VBSs to vinculin's Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculin's Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and alpha-actinin binding in focal adhesions versus adherens junctions, respectively.

FoxO1a-cyclic GMP-dependent Kinase I Interactions Orchestrate Myoblast Fusion

Molecular and Cellular Biology. Sep, 2005  |  Pubmed ID: 16107711

The regulatory circuits that orchestrate mammalian myoblast cell fusion during myogenesis are poorly understood. The transcriptional activity of FoxO1a directly regulates this process, yet the molecular mechanisms governing FoxO1a activity during muscle cell differentiation remain unknown. Here we show an autoregulatory loop in which FoxO1a directly activates transcription of the cyclic GMP-dependent protein kinase I (cGKI) gene and where the ensuing cGKI activity phosphorylates FoxO1a and abolishes its DNA binding activity. These findings establish the FoxO1a-to-cGKI pathway as a novel feedback loop that allows the precise tuning of myoblast fusion. Interestingly, this pathway appears to operate independently of muscle cell differentiation programs directed by myogenic transcription factors.

FOXO1a Acts As a Selective Tumor Suppressor in Alveolar Rhabdomyosarcoma

The Journal of Cell Biology. Sep, 2005  |  Pubmed ID: 16157701

Rhabdomyosarcoma (RMS), the most common pediatric soft-tissue sarcoma, has two major histological subtypes: embryonal RMS (ERMS), which has a favorable prognosis, and alveolar RMS (ARMS), which has a poor outcome. Although both forms of RMS express muscle cell-specific markers, only ARMS cells express PAX3-FOXO1a or PAX7-FOXO1a chimeric proteins. In mice, Pax3 and Pax7 play key roles in muscle cell development and differentiation, and FoxO1a regulates myoblast differentiation and fusion; thus, the aberrant regulation of these proteins may contribute to the development of ARMS. In this paper, we report that FOXO1a is not expressed in primary ARMS tumors or ARMS-derived tumor cell lines and that restoration of FOXO1a expression in ARMS cells is sufficient to induce cell cycle arrest and apoptosis. Strikingly, the effects of FOXO1a are selective, as enforced expression of FOXO1a in ERMS-derived tumor cell lines had no effect. Furthermore, FOXO1a induced apoptosis in ARMS by directly activating the transcription of caspase-3. We conclude that FOXO1a is a potent and specific tumor suppressor in ARMS, suggesting that agents that restore or augment FOXO1a activity may be effective as ARMS therapeutics.

The Vinculin Binding Sites of Talin and Alpha-actinin Are Sufficient to Activate Vinculin

The Journal of Biological Chemistry. Mar, 2006  |  Pubmed ID: 16407299

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.

Shigella Applies Molecular Mimicry to Subvert Vinculin and Invade Host Cells

The Journal of Cell Biology. Nov, 2006  |  Pubmed ID: 17088427

Shigella flexneri, the causative agent of bacillary dysentery, injects invasin proteins through a type III secretion apparatus upon contacting the host cell, which triggers pathogen internalization. The invasin IpaA is essential for S. flexneri pathogenesis and binds to the cytoskeletal protein vinculin to facilitate host cell entry. We report that IpaA harbors two vinculin-binding sites (VBSs) within its C-terminal domain that bind to and activate vinculin in a mutually exclusive fashion. Only the highest affinity C-terminal IpaA VBS is necessary for efficient entry and cell-cell spread of S. flexneri, whereas the lower affinity VBS appears to contribute to vinculin recruitment at entry foci of the pathogen. Finally, the crystal structures of vinculin in complex with the VBSs of IpaA reveal the mechanism by which IpaA subverts vinculin's functions, where S. flexneri utilizes a remarkable level of molecular mimicry of the talin-vinculin interaction to activate vinculin. Mimicry of vinculin's interactions may therefore be a general mechanism applied by pathogens to infect the host cell.

FOXO1a Acts As a Selective Tumor Suppressor in Alveolar Rhabdomyosarcoma

The Journal of Cell Biology. May, 2007  |  Pubmed ID: 17485494

Species-wide Distribution of Highly Polymorphic Minisatellite Markers Suggests Past and Present Genetic Exchanges Among House Mouse Subspecies

Genome Biology. 2007  |  Pubmed ID: 17501990

Four hypervariable minisatellite loci were scored on a panel of 116 individuals of various geographical origins representing a large part of the diversity present in house mouse subspecies. Internal structures of alleles were determined by minisatellite variant repeat mapping PCR to produce maps of intermingled patterns of variant repeats along the repeat array. To reconstruct the genealogy of these arrays of variable length, the specifically designed software MS_Align was used to estimate molecular divergences, graphically represented as neighbor-joining trees.

A Highly Polymorphic Meiotic Recombination Mouse Hot Spot Exhibits Incomplete Repair

Molecular and Cellular Biology. Oct, 2007  |  Pubmed ID: 17709383

The recent mapping of recombination hot spots in the human genome has demonstrated that crossover is a nonrandom process that occurs at well-defined positions along chromosomes. However, the mechanisms that direct hot-spot turnover in complex mammalian genomes are poorly understood. Analyses of the human genome are impaired by the inability to genetically dissect and molecularly manipulate recombinogenic regions to test their roles in regulating hot spots. Here, using the BXD recombinant inbred strains as a crossover library, three new recombination hot spots have been identified on mouse chromosome 19. Analyses of a highly polymorphic recombination hot spot (HS22) revealed that approximately 4% of recombinant molecules display complex and incomplete repair with discontinuous conversion tracts, as well as persistent heteroduplex DNA at crossover sites in mature spermatozoa. Also, sequence analysis of the wild house mouse revealed instability at the center of this hot spot. This suggests that complete repair is not required for completion of mammalian meiosis, a scenario that leaves duplex DNA containing mismatches at crossover sites.

Genetic Analysis of Variation in Human Meiotic Recombination

PLoS Genetics. Sep, 2009  |  Pubmed ID: 19763160

The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31) were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1), results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.

Anatomy of Mouse Recombination Hot Spots

Nucleic Acids Research. Apr, 2010  |  Pubmed ID: 20081202

Genome-wide analyses have suggested thousands of meiotic recombination hot spots across mammalian genomes. However, very few hot spots have been directly analyzed at a sub-kb scale for crossover (CO) activity. Using recombinant inbred strains as a CO library, here we report the identification and detailed characterization of seven new meiotic hot spots on mouse chromosome 19, more than doubling the number of currently available mouse hot spots. Although a shared feature is the narrow 1.5-2.5-kb width of these recombinogenic sites, these analyses revealed that hot spots have diverse sequence attributes and distinct symmetric and asymmetric CO profiles. Interestingly, CO molecules with discontinuous conversion tracts are commonly observed, contrasting with those found in human. Furthermore, unlike human hot spots, those present in the mouse do not necessarily have a quasi-normal CO distribution but harbor CO repulsion zones within recombinogenic cores. We propose a model where local chromatin landscape directs these repulsion zones.

Nucleosome Occupancy Landscape and Dynamics at Mouse Recombination Hotspots

EMBO Reports. Jul, 2010  |  Pubmed ID: 20508641

During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double-strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover-free zones in recombinogenic domains. Collectively, these high-resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.