Showing papers in "Chromosome Research in 2013"

Epigenetic regulation by long noncoding RNAs in plants.

Abstract: Many eukaryotes, including plants, produce a large number of long noncoding RNAs (lncRNAs). Growing number of lncRNAs are being reported to have regulatory roles in various developmental processes. Emerging mechanisms underlying the function of lncRNAs indicate that lncRNAs are versatile regulatory molecules. They function as potent cis- and trans-regulators of gene expression, including the formation of modular scaffolds that recruit chromatin-modifying complexes to target chromatin. LncRNAs have also been reported in plants. Here, we describe our current understanding on potential roles of lncRNA in plants.

RNAi function, diversity, and loss in the fungal kingdom

Abstract: RNAi is conserved and has been studied in a broad cross-section of the fungal kingdom, including Neurospora crassa, Schizosaccharomyces pombe, Cryptococcus neoformans, and Mucor circinelloides. And yet well known species, including the model yeast Saccharomyces cerevisiae and the plant pathogen Ustilago maydis, have lost RNAi, providing insights and opportunities to illuminate benefits conferred both by the presence of RNAi and its loss. Some of the earliest studies of RNAi were conducted in Neurospora, contemporaneously with the elucidation of RNAi in Caenorhabditis elegans. RNAi is a key epigenetic mechanism for maintaining genomic stability and integrity, as well as to defend against viruses, and given its ubiquity was likely present in the last eukaryotic common ancestor. In this review, we describe the diversity of RNAi mechanisms found in the fungi, highlighting recent work in Neurospora, S. pombe, and Cryptococcus. Finally, we consider frequent, independent losses of RNAi in diverse fungal lineages and both review and speculate on evolutionary forces that may drive the losses or result therefrom.

A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone spinifera, reveals an intriguing sex chromosome conservation in Trionychidae

Abstract: Reptiles display a wide diversity of sex-determining mechanisms ranging from temperature-dependent sex determination (TSD) to genotypic sex determination (GSD) with either male (XY) or female (ZW) heterogamety. Despite this astounding variability, the origin, structure, and evolution of sex chromosomes remain poorly understood. In turtles, TSD is purportedly ancestral while GSD arose multiple times independently. Here we test whether independent (XY or ZW) or morphologically divergent heterogametic sex chromosome systems evolved in tryonichids (Cryptodira) using the GSD spiny softshell turtle, Apalone spinifera, a species with previously unidentified sex chromosomes. A female-specific signal from comparative genomic hybridization (CGH) was detected in a Giemsa/4′,6-diamidino-2-phenylindole faint portion of a microchromosome, indicating the presence of a ZZ/ZW system in A. spinifera. In situ hybridization of a fluorescently labeled 18S rRNA probe identified a large nucleolar organizer region block in the female-specific region of the W (co-localizing with the female-specific CGH signal) and a smaller block on the Z. The heteromorphic ZZ/ZW micro-sex chromosome system detected here is identical to that found in another tryonichid, the Chinese softshell turtle Pelodiscus sinensis, from which A. spinifera diverged ∼95 million years ago. These results reveal a striking sex chromosome conservation in tryonichids, compared to the divergent sex chromosome morphology observed among younger XX/XY systems in pleurodiran turtles. Our findings highlight the need to understand the drivers behind sex chromosome lability and conservation in different lineages and contribute to our knowledge of sex chromosome evolution in reptiles and vertebrates.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

Abstract: The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes.

Molecular cytogenetic characterization of a new wheat-rye 4R chromosome translocation line resistant to powdery mildew

Abstract: Rye is an important and valuable gene resource for wheat improvement. However, due to extensive growing of cultivars with disease resistance genes from short arm of rye chromosome 1R and coevolution of pathogen virulence and host resistance, these cultivars successively lost resistance to pathogens. Identification and deployment of new resistance gene sources in rye are, therefore, of especial importance and urgency. A new wheat–rye line, designated as WR41-1, was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. It was proved to be a new wheat–rye T4BL·4RL and T7AS·4RS translocation line using sequential genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), and expressed sequence tag-simple sequence repeat (EST-SSR) marker analysis. WR41-1 showed high levels of resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 13 of 23 Bgt isolates tested at the seedling stage. According to its resistant pattern to 23 different Bgt isolates, WR41-1 may possess new gene(s) for resistance to powdery mildew, which differed from previously identified and known powdery mildew genes from rye (Pm7, Pm8, Pm17, and Pm20). In addition, WR41-1 was cytologically stable, had a desirable fertility, and is expected to be useful in wheat improvement.

EdU induces DNA damage response and cell death in mESC in culture

Abstract: Recently, a novel DNA replication precursor analogue called 5-ethynyl-2′-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis

Abstract: Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a “dicentric” chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

Epigenetics of eu- and heterochromatin in inverted and conventional nuclei from mouse retina

Abstract: To improve light propagation through the retina, the rod nuclei of nocturnal mammals are uniquely changed compared to the nuclei of other cells. In particular, the main classes of chromatin are segregated in them and form regular concentric shells in order; inverted in comparison to conventional nuclei. A broad study of the epigenetic landscape of the inverted and conventional mouse retinal nuclei indicated several differences between them and several features of general interest for the organization of the mammalian nuclei. In difference to nuclei with conventional architecture, the packing density of pericentromeric satellites and LINE-rich chromatin is similar in inverted rod nuclei; euchromatin has a lower packing density in both cases. A high global chromatin condensation in rod nuclei minimizes the structural difference between active and inactive X chromosome homologues. DNA methylation is observed primarily in the chromocenter, Dnmt1 is primarily associated with the euchromatic shell. Heterochromatin proteins HP1-alpha and HP1-beta localize in heterochromatic shells, whereas HP1-gamma is associated with euchromatin. For most of the 25 studied histone modifications, we observed predominant colocalization with a certain main chromatin class. Both inversions in rod nuclei and maintenance of peripheral heterochromatin in conventional nuclei are not affected by a loss or depletion of the major silencing core histone modifications in respective knock-out mice, but for different reasons. Maintenance of peripheral heterochromatin appears to be ensured by redundancy both at the level of enzymes setting the epigenetic code (writers) and the code itself, whereas inversion in rods rely on the absence of the peripheral heterochromatin tethers (absence of code readers).

Karyotype evolution in monitor lizards: cross-species chromosome mapping of cDNA reveals highly conserved synteny and gene order in the Toxicofera clade

Abstract: The water monitor lizard (Varanus salvator macromaculatus (VSA), Platynota) has a chromosome number of 2n = 40: its karyotype consists of 16 macrochromosomes and 24 microchromosomes To delineate the process of karyotype evolution in V salvator macromaculatus, we constructed a cytogenetic map with 86 functional genes and compared it with those of the butterfly lizard (Leiolepis reevesii rubritaeniata (LRE); 2n = 36) and Japanese four-striped rat snake (Elaphe quadrivirgata (EQU); 2n = 36), members of the Toxicofera clade The syntenies and gene orders of macrochromosomes were highly conserved between these species except for several chromosomal rearrangements: eight pairs of VSA macrochromosomes and/or chromosome arms exhibited homology with six pairs of LRE macrochromosomes and eight pairs of EQU macrochromosomes Furthermore, the genes mapped to microchromosomes of three species were all located on chicken microchromosomes or chromosome 4p No reciprocal translocations were found in the species, and their karyotypic differences were caused by: low frequencies of interchromosomal rearrangements, such as tandem fusions, or centric fissions/fusions between macrochromosomes and between macro- and microchromosomes; and intrachromosomal rearrangements, such as paracentric inversions or centromere repositioning The chromosomal rearrangements that occurred in macrochromosomes of the Varanus lineage were also identified through comparative cytogenetic mapping of V salvator macromaculatus and V exanthematicus Morphologic differences in chromosomes 6–8 between the two species could have resulted from pericentric inversion or centromere repositioning

Molecular cytogenetic map of the central bearded dragon, Pogona vitticeps (Squamata: Agamidae).

Abstract: Reptiles, as the sister group to birds and mammals, are particularly valuable for comparative genomic studies among amniotes. The Australian central bearded dragon (Pogona vitticeps) is being developed as a reptilian model for such comparisons, with whole-genome sequencing near completion. The karyotype consists of 6 pairs of macrochromosomes and 10 pairs microchromosomes (2n = 32), including a female heterogametic ZW sex microchromosome pair. Here, we present a molecular cytogenetic map for P. vitticeps comprising 87 anchor bacterial artificial chromosome clones that together span each macro- and microchromosome. It is the first comprehensive cytogenetic map for any non-avian reptile. We identified an active nucleolus organizer region (NOR) on the sub-telomeric region of 2q by mapping 18S rDNA and Ag-NOR staining. We identified interstitial telomeric sequences in two microchromosome pairs and the W chromosome, indicating that microchromosome fusion has been a mechanism of karyotypic evolution in Australian agamids within the last 21 to 19 million years. Orthology searches against the chicken genome revealed an intrachromosomal rearrangement of P. vitticeps 1q, identified regions orthologous to chicken Z on P. vitticeps 2q, snake Z on P. vitticeps 6q and the autosomal microchromosome pair in P. vitticeps orthologous to turtle Pelodiscus sinensis ZW and lizard Anolis carolinensis XY. This cytogenetic map will be a valuable reference tool for future gene mapping studies and will provide the framework for the work currently underway to physically anchor genome sequences to chromosomes for this model Australian squamate.

Interstitial telomeric repeats are enriched in the centromeres of chromosomes in Solanum species

Abstract: Interstitial telomeric repeats (ITRs) were reported in a number of animal and plant species. Most ITRs are organized as short tandem arrays and are likely evolutionary relics derived from chromosomal rearrangements and DNA repairs. However, megabase-sized ITR arrays were reported in Solanum species. Here, we report a fluorescence in situ hybridization (FISH) survey of ITRs in all representative diploid Solanum species, including potato, tomato, and eggplant. FISH revealed massive amplification of ITRs in the centromeric regions of chromosomes from the Solanum species containing the B and P genomes. A significant proportion of the ITR FISH signals was mapped within the primary constrictions of the pachytene chromosomes of Solanum pinnatisectum (B genome). In addition, some ITR sites overlapped with St49, a satellite repeat enriched in centromeric DNA sequences associated with CENH3 nucleosomes, in both A and B genome Solanum species. These results show that some ITR subfamilies have been amplified and invaded in the functional centromeres of chromosomes in Solanum species.

High-throughput sequencing of a single chromosome: a moth W chromosome

Abstract: Y and W chromosomes have mostly been excluded from whole genome sequencing projects. Due to the high amount of repetitive sequences they are 'difficult' to assemble and therefore need special treatment in the form of, e.g. adapted assembly programs, a range of different libraries, and accurate maps, if possible. A minimum requirement for these approaches is pure template DNA. We therefore microdissected the W chromatin of highly polyploid cells from the flour moth, Ephestia kuehniella, and used Roche/454 and Sanger sequencing to generate 72.6 Mbp of DNA sequence. Nominal coverage was 4.3× of the 16.7 Mbp of W chromosomal DNA. We used these data to assess the genetic content of the W chromosome. This approach allowed us to determine constituent families of transposable elements, microsatellites, and recent insertion sites of mitochondrial DNA. However, no conventional protein-coding gene has yet been found. The sequence collection is a rich source for the definition of W-specific PCR markers and the reconstruction of W chromosome loci, as a step towards full reconstruction of the chromosome.

High chromosome variability and the presence of multivalent associations in buthid scorpions

Abstract: In this study, we investigated the mitotic and meiotic chromosomes of 11 Buthidae scorpion species, belonging to three genera (Ananteris, Rhopalurus and Tityus), to obtain detailed knowledge regarding the mechanisms underlying the intraspecific and/or interspecific diversity of chromosome number and the origin of the complex chromosome associations observed during meiosis. The chromosomes of all species did not exhibit a localised centromere region and presented synaptic and achiasmatic behaviour during meiosis I. Spermatogonial and/or oogonial metaphase cells of these buthids showed diploid numbers range from 2n = 6 to 2n = 28. In most species, multivalent chromosome associations were observed in pachytene and postpachytene nuclei. Moreover, intraspecific variability associated with the presence or absence of chromosome chains and the number of chromosomes in the complex meiotic configurations was observed in some species of these three genera. Silver-impregnated cells revealed that the number and location of nucleolar organiser regions (NORs) remained unchanged despite extensive chromosome variation; notably, two NORs located on the terminal or subterminal chromosome regions were commonly observed for all species. C-banded and fluorochrome-stained cells showed that species with conspicuous blocks of heterochromatin exhibited the lowest rate of chromosomal rearrangement. Based on the investigation of mitotic and meiotic cells, we determined that the intraspecific variability occurred as a consequence of fission/fusion-type chromosomal rearrangements in Ananteris and Tityus species and reciprocal translocation in Rhopalurus species. Furthermore, we verified that individuals presenting the same diploid number differ in structural chromosome organisation, giving rise to intraspecific differences of chromosome association in meiotic cells (bivalent-like elements or chromosome chains).

Small RNAs, big impact: small RNA pathways in transposon control and their effect on the host stress response

Abstract: Transposons are mobile genetic elements that are a major constituent of most genomes. Organisms regulate transposable element expression, transposition, and insertion site preference, mitigating the genome instability caused by uncontrolled transposition. A recent burst of research has demonstrated the critical role of small non-coding RNAs in regulating transposition in fungi, plants, and animals. While mechanistically distinct, these pathways work through a conserved paradigm. The presence of a transposon is communicated by the presence of its RNA or by its integration into specific genomic loci. These signals are then translated into small non-coding RNAs that guide epigenetic modifications and gene silencing back to the transposon. In addition to being regulated by the host, transposable elements are themselves capable of influencing host gene expression. Transposon expression is responsive to environmental signals, and many transposons are activated by various cellular stresses. TEs can confer local gene regulation by acting as enhancers and can also confer global gene regulation through their non-coding RNAs. Thus, transposable elements can act as stress-responsive regulators that control host gene expression in cis and trans.

DNA abandonment and the mechanisms of uniparental inheritance of mitochondria and chloroplasts.

Abstract: For most eukaryotic organisms, the nuclear genomes of both parents are transmitted to the progeny following biparental inheritance. For mitochondria and chloroplasts, however, uniparental inheritance (UPI) is frequently observed. The maternal mode of inheritance for mitochondria in animals can be nearly absolute, suggesting an adaptive advantage for UPI. In other organisms, however, the mode of inheritance for mitochondria and chloroplasts can vary greatly even among strains of a species. Here, I review the data on the transmission of organellar DNA (orgDNA) from parent to progeny and the structure, copy number, and stability of orgDNA molecules. I propose that UPI is an incidental by-product of DNA abandonment, a process that lowers the metabolic cost of orgDNA repair.

Mapping nonrecombining regions in barley using multicolor FISH

Abstract: Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.

Hypermorphic expression of centromeric retroelement-encoded small RNAs impairs CENP-A loading

Abstract: The proper functioning of centromeres requires a complex cascade of epigenetic events involving chromatin and kinetochore assembly; however, the precise mechanism by which this cascade proceeds is unknown. The pivotal event during kinetochore formation is the "loading," or deposition, of CENP-A. This histone H3 variant is specific to centromeres and replaces conventional H3 in centromeric chromatin. Failure to load CENP-A into mammalian centromeres in late telophase/early G1 of the cell cycle leads to malsegregation and cell division defects in subsequent cell cycles. Mounting evidence supports the hypothesis that an RNA component is involved, although how RNAs participate in centromere formation in mammals has remained unknown. Using the marsupial model, the tammar wallaby, we show that centromeric retroelements produce small RNAs and that hypermorphic expression of these centromeric small RNAs results in disruption of CENP-A localization. We propose that tight regulation of the processing of this new class of small RNAs, crasiRNAs, is an integral component of the epigenetic framework necessary for centromere establishment.

Chromatin-associated ncRNA activities.

Abstract: RNA transcripts that do not code for proteins have been long known to lie at the heart of many biological processes, such as splicing and translation. Yet their full potential has only been appreciated recently and non-coding RNAs (ncRNAs) are now attracting increasing attention. Pioneering work in yeast and plant systems has revealed that non-coding RNAs can have a major influence on the deposition of histone and DNA modifications. This can introduce heritable variation into gene expression and, thus, be the basis of epigenetic phenomena. Mechanistically, such processes have been studied extensively in the fission yeast Schizosaccharomyces pombe, providing an important conceptual framework for possible modes of action of ncRNAs also in other organisms. In this review, we highlight mechanistic insights into chromatin-associated ncRNA activities gained from work with fission yeast, and we draw parallels to studies in other eukaryotes that indicate evolutionary conservation.

Functional insights into the role of nuclear-retained long noncoding RNAs in gene expression control in mammalian cells

Abstract: The mammalian genome harbors thousands of long noncoding RNA (lncRNA) genes. Recent studies have indicated the involvement of several of these lncRNAs in the regulation of gene expression. lncRNAs play crucial roles in various biological processes ranging from epigenetic gene regulation, transcriptional control,to post-transcriptional regulation. lncRNAs are localized in various subcellular compartments, and major proportion of these are retained in the cell nucleus and could be broadly classified as nuclear-retained lncRNAs (nrRNAs). Based on the identified functions,members of the nrRNAs execute diverse roles, including providing architectural support to the hierarchical subnuclear organization and influencing the recruitment of chromatin modifier factors to specific chromatin sites. In this review, we will summarize the recently described roles of mammalian nrRNAs in controlling gene expression by influencing chromatin organization, transcription,pre-mRNA processing, nuclear organization, and their involvement in disease.

FISH with whole chromosome and telomeric probes demonstrates huge karyotypic reorganization with ITS between two species of Oryzomyini (Sigmodontinae, Rodentia): Hylaeamys megacephalus probes on Cerradomys langguthi karyotype.

Abstract: Rodentia comprises 42 % of living mammalian species. The taxonomic identification can be difficult, the number of species currently known probably being underestimated, since many species show only slight morphological variations. Few studies surveyed the biodiversity of species, especially in the Amazon region. Cytogenetic studies show great chromosomal variability in rodents, with diploid numbers ranging from 10 to 102, making it difficult to find chromosomal homologies by comparative G banding. Chromosome painting is useful, but only a few species of rodents have been studied by this technique. In this study, we sorted whole chromosome probes by fluorescence-activated cell sorting from two Hylaeamys megacephalus individuals, an adult female (2n = 54) and a fetus (2n = 50). We made reciprocal chromosome painting between these karyotypes and cross-species hybridization on Cerradomys langguthi (2n = 46). Both species belong to the tribe Oryzomyini (Sigmodontinae), which is restricted to South America and were collected in the Amazon region. Twenty-four chromosome-specific probes from the female and 25 from the fetus were sorted. Reciprocal chromosome painting shows that the karyotype of the fetus does not represent a new cytotype, but an unbalanced karyotype with multiple rearrangements. Cross-species hybridization of H. megacephalus probes on metaphases of C. langguthi shows that 11 chromosomes of H. megacephalus revealed conserved synteny, 10 H. megacephalus probes hybridized to two chromosomal regions and three hybridized to three regions. Associations were observed on chromosomes pairs 1–4 and 11. Fluorescence in situ hybridization with a telomeric probe revealed interstitial regions in three pairs (1, 3, and 4) of C. langguthi chromosomes. We discuss the genomic reorganization of the C. langguthi karyotype.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

Abstract: The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Asymmetric distribution of histones during Drosophila male germline stem cell asymmetric divisions.

Abstract: It has long been known that epigenetic changes are inheritable. However, except for DNA methylation, little is known about the molecular mechanisms of epigenetic inheritance. Many types of stem cells undergo asymmetric cell divisions to generate self-renewed stem cells and daughter cells committed for differentiation. Still, whether and how stem cells retain their epigenetic memory remain questions to be elucidated. During the asymmetric division of Drosophila male germline stem cell (GSC), our recent studies revealed that the preexisting histone 3 (H3) are selectively segregated to the GSC, whereas newly synthesized H3 deposited during DNA replication are enriched in the differentiating daughter cell. We propose a two-step model to explain this asymmetric histone distribution. First, prior to mitosis, preexisting histones and newly synthesized histones are differentially distributed at two sets of sister chromatids. Next, during mitosis, the set of sister chromatids that mainly consist of preexisting histones are segregated to GSCs, while the other set of sister chromatids enriched with newly synthesized histones are partitioned to the daughter cell committed for differentiation. In this review, we apply current knowledge about epigenetic inheritance and asymmetric cell division to inform our discussion of potential molecular mechanisms and the cellular basis underlying this asymmetric histone distribution pattern. We will also discuss whether this phenomenon contributes to the maintenance of stem cell identity and resetting chromatin structure in the other daughter cell for differentiation.

Chromosomal speciation in mice: a cytogenetic analysis of recombination.

Abstract: Within species, populations differing by chromosomal rearrangements (“chromosomal races”) may become reproductively isolated in association with reduced hybrid fertility due to meiotic aberrations. Speciation is also possible if hybridizing chromosomal races accumulate genetic differences because of reduced meiotic recombination in the heterozygous configuration in hybrids. Here, we examine recombination in pure races and hybrids within a model system for chromosomal speciation: the hybridization of the Poschiavo (CHPO) and Upper Valtellina (IUVA) chromosomal races of house mouse in Upper Valtellina, Italy. These races differ by Robertsonian fusions/whole-arm reciprocal translocations, such that hybrids produce a pentavalent meiotic configuration. We determined the number and position of the recombination points (using an antibody against the MutL homolog 1 [MLH1] protein) on synaptonemal complexes at pachytene in laboratory-reared CHPO, IUVA, and hybrid males, analyzing at least 112 spermatocytes per karyotypic group, up to a total of 534 spermatocytes. The mean ± standard deviation numbers of MLH1 foci per spermatocyte were 22.2 ± 3.2, 20.1 ± 2.9, 20.7 ± 2.3, and 21.9 ± 2.9 for CHPO, IUVA, CHPO × IUVA, and IUVA × CHPO, respectively. Altogether, 10,146 chromosome arms were examined, allowing multiple comparisons. Overall, recombination events were more frequently distal than proximal or interstitial. The average number of proximal MLH1 foci per chromosome arm decreased going from telocentric to metacentric bivalents to pentavalents (when present), which (together with other factors) influenced the average number of MLH1 foci per cell between CHPO, IUVA, and hybrid mice. The low frequency of proximal recombination in pentavalents of CHPO–IUVA hybrids may promote reproductive isolation between the CHPO and IUVA races, when coupled with reduced hybrid unfitness.

Directional genomic hybridization for chromosomal inversion discovery and detection.

Abstract: Chromosomal rearrangements are a source of structural variation within the genome that figure prominently in human disease, where the importance of translocations and deletions is well recognized. In principle, inversions—reversals in the orientation of DNA sequences within a chromosome—should have similar detrimental potential. However, the study of inversions has been hampered by traditional approaches used for their detection, which are not particularly robust. Even with significant advances in whole genome approaches, changes in the absolute orientation of DNA remain difficult to detect routinely. Consequently, our understanding of inversions is still surprisingly limited, as is our appreciation for their frequency and involvement in human disease. Here, we introduce the directional genomic hybridization methodology of chromatid painting—a whole new way of looking at structural features of the genome—that can be employed with high resolution on a cell-by-cell basis, and demonstrate its basic capabilities for genome-wide discovery and targeted detection of inversions. Bioinformatics enabled development of sequence- and strand-specific directional probe sets, which when coupled with single-stranded hybridization, greatly improved the resolution and ease of inversion detection. We highlight examples of the far-ranging applicability of this cytogenomics-based approach, which include confirmation of the alignment of the human genome database and evidence that individuals themselves share similar sequence directionality, as well as use in comparative and evolutionary studies for any species whose genome has been sequenced. In addition to applications related to basic mechanistic studies, the information obtainable with strand-specific hybridization strategies may ultimately enable novel gene discovery, thereby benefitting the diagnosis and treatment of a variety of human disease states and disorders including cancer, autism, and idiopathic infertility.

A multitasking Argonaute: exploring the many facets of C. elegans CSR-1

Abstract: While initial studies of small RNA-mediated gene regulatory pathways focused on the cytoplasmic functions of such pathways, identifying roles for Argonaute/small RNA pathways in modulating chromatin and organizing the genome has become a topic of intense research in recent years. Nuclear regulatory mechanisms for Argonaute/small RNA pathways appear to be widespread, in organisms ranging from plants to fission yeast, Caenorhabditis elegans to humans. As the effectors of small RNA-mediated gene regulatory pathways, Argonaute proteins guide the chromatin-directed activities of these pathways. Of particular interest is the C. elegans Argonaute, chromosome segregation and RNAi deficient (CSR-1), which has been implicated in such diverse functions as organizing the holocentromeres of worm chromosomes, modulating germline chromatin, protecting the genome from foreign nucleic acid, regulating histone levels, executing RNAi, and inhibiting translation in conjunction with Pumilio proteins. CSR-1 interacts with small RNAs known as 22G-RNAs, which have complementarity to 25 % of the protein coding genes. This peculiar Argonaute is the only essential C. elegans Argonaute out of 24 family members in total. Here, we summarize the current understanding of CSR-1 functions in the worm, with emphasis on the chromatin-directed activities of this ever-intriguing Argonaute.

Long nonoding RNAs in the X-inactivation center.

Abstract: The X-inactivation center is a hotbed of functional long noncoding RNAs in eutherian mammals. These RNAs are thought to help orchestrate the epigenetic transcriptional states of the two X-chromosomes in females as well as of the single X-chromosome in males. To balance X-linked gene expression between the sexes, females undergo transcriptional silencing of most genes on one of the two X-chromosomes in a process termed X-chromosome inactivation. While one X-chromosome is inactivated, the other X-chromosome remains active. Moreover, with a few notable exceptions, the originally established epigenetic transcriptional profiles of the two X-chromosomes is maintained as such through many rounds of cell division, essentially for the life of the organism. The stable and divergent transcriptional fates of the two X-chromosomes, despite residing in a shared nucleoplasm, make X-inactivation a paradigm of epigenetic transcriptional regulation. Originally proposed in 1961 by Mary Lyon, the X-inactivation hypothesis has been validated through much experimentation. In the last 25 years, the discovery and functional characterization has firmly established X-linked long noncoding RNAs as key players in choreographing X-chromosome inactivation.

Chromosome conformation capture-on-chip analysis of long-range cis-interactions of the SOX9 promoter.

Abstract: Evolutionarily conserved transcription factor SOX9 is essential for the differentiation of chondrocytes and the development of testes. Heterozygous point mutations and genomic deletions involving SOX9 lead to campomelic dysplasia (CD), a skeletal malformation syndrome often associated with sex reversal. Chromosomal rearrangements with breakpoints mapping up to 1.6 Mb up- and downstream to SOX9, and likely disrupting its distant cis-regulatory elements, have been described in patients with milder forms of CD. Based on the location of these aberration breakpoints, four clusters upstream of SOX9 have been defined. Interestingly, we found that each of these intervals overlaps a gene encoding long noncoding RNA (lncRNA), suggesting that lncRNAs may contribute to long-range regulation of SOX9 expression. One of the four upstream regions, RevSex (517–595 kb 5′ to SOX9), is associated with sex reversal, and was suggested to harbor a testis-specific and sex-determining enhancer. Another sex-determining interval was mapped to a gene desert >1.3 Mb downstream of SOX9. We have performed chromosome conformation capture-on-chip (4C) analysis in Sertoli cells and lymphoblasts to verify the proposed long-range interactions of the SOX9 promoter and to identify potential novel regulatory elements that might be responsible for sex reversal in patients with CD. We identified several novel potentially cis-interacting regions both up- and downstream to SOX9, with some of them overlapping lncRNA genes. Our data point to lncRNAs as likely mediators of some of these regulatory interactions.

An assessment of karyotype restructuring in the neoallotetraploid Tragopogon miscellus (Asteraceae)

Abstract: Tragopogon miscellus and Tragopogon mirus are two rare examples of allopolyploids that have formed recently in nature. Molecular cytogenetic studies have revealed chromosome copy number variation and intergenomic translocations in both allotetraploids. Due to a lack of interstitial chromosome markers, there remained the possibility of additional karyotype restructuring in these neopolyploids, via intrachromosomal and intragenomic rearrangements. To address this issue, we searched for additional high-copy tandem repeats in genomic sequences of the diploid progenitor species—Tragopogon dubius, Tragopogon pratensis and Tragopogon porrifolius—for application to the chromosomes of the allotetraploids. Eight novel repeats were localised by fluorescence in situ hybridisation (FISH) in the diploids; one of these repeats, TTR3, provided interstitial coverage. TTR3 was included in a cocktail with other previously characterised probes, producing better-resolved karyotypes for the three diploids. The cocktail was then used to test a hypothesis of karyotype restructuring in the recent allotetraploid T. miscellus by comparing repeat distributions to its diploid progenitors, T. dubius and T. pratensis. Five individuals of T. miscellus were selected from across the range of karyotypic variation previously observed in natural populations. FISH signal distributions mostly matched those observed in the diploid progenitors, with the exception of several losses or gains of signal at chromosomal subtermini and previously noted intergenomic translocations. Thus, in T. miscellus, we find most changes restricted to the subterminal regions, and although some were recurrent, none of the changes were common to all individuals analysed. We consider these findings in relation to the gene loss reported previously for T. miscellus.

Transcription and ncRNAs: at the cent(rome)re of kinetochore assembly and maintenance.

Abstract: Centromeres are sites of chromosomal spindle attachment during mitosis and meiosis. Centromeres are defined, in part, by a distinct chromatin landscape in which histone H3 is replaced by the conserved histone H3 variant, CENP-A. Sequences competent for centromere formation and function vary among organisms and are typically composed of repetitive DNA. It is unclear how such diverse genomic signals are integrated with the epigenetic mechanisms that govern CENP-A incorporation at a single locus on each chromosome. Recent work highlights the intriguing possibility that the transcriptional properties of centromeric core DNA contribute to centromere identity and maintenance through cell division. Moreover, core-derived noncoding RNAs (ncRNAs) have emerged as active participants in the regulation and control of centromere activity in plants and mammals. This paper reviews the transcriptional properties of eukaryotic centromeres and discusses the known roles of core-derived ncRNAs in chromatin integrity, kinetochore assembly, and centromere activity.

Long noncoding RNA-based chromatin control of germ cell differentiation: a yeast perspective

Abstract: Germ cell differentiation, the cellular process by which a diploid progenitor cell produces by meiotic divisions haploid cells, is conserved from the unicellular yeasts to mammals. Over the recent years, yeast germ cell differentiation process has proven to be a powerful biological system to identify and study several long noncoding RNAs (lncRNAs) that play a central role in regulating cellular differentiation by acting directly on chromatin. Remarkably, in the well-studied budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, the lncRNA-based chromatin regulations of germ cell differentiation are quite different. In this review, we present an overview of these regulations by focusing on the mechanisms and their respective functions both in S. cerevisiae and in S. pombe. Part of these lncRNA-based chromatin regulations may be conserved in other eukaryotes and play critical roles either in the context of germ cell differentiation or, more generally, in the development of multicellular organisms.