The architecture and evolution of cancer neochromosomes.

We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corrosion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.

Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells.

Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line "age," given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.

The evolutionary life cycle of the resilient centromere.

The centromere is a chromosomal structure that is essential for the accurate segregation of replicated eukaryotic chromosomes to daughter cells. In most centromeres, the underlying DNA is principally made up of repetitive DNA elements, such as tandemly repeated satellite DNA and retrotransposable elements. Paradoxically, for such an essential genomic region, the DNA is rapidly evolving both within and between species. In this review, we show that the centromere locus is a resilient structure that can undergo evolutionary cycles of birth, growth, maturity, death and resurrection. The birth phase is highlighted by examples in humans and other organisms where centromere DNA deletions or chromosome rearrangements can trigger the epigenetic assembly of neocentromeres onto genomic sites without typical features of centromere DNA. In addition, functional centromeres can be generated in the laboratory using various methodologies. Recent mapping of the foundation centromere mark, the histone H3 variant CENP-A, onto near-complete genomes has uncovered examples of new centromeres which have not accumulated centromere repeat DNA. During the growth period of the centromere, repeat DNA begins to appear at some, but not all, loci. The maturity stage is characterised by centromere repeat accumulation, expansions and contractions and the rapid evolution of the centromere DNA between chromosomes of the same species and between species. This stage provides inherent centromere stability, facilitated by repression of gene activity and meiotic recombination at and around the centromeres. Death to a centromere can result from genomic instability precipitating rearrangements, deletions, accumulation of mutations and the loss of essential centromere binding proteins. Surprisingly, ancestral centromeres can undergo resurrection either in the field or in the laboratory, via as yet poorly understood mechanisms. The underlying principle for the preservation of a centromeric evolutionary life cycle is to provide resilience and perpetuity for the all-important structure and function of the centromere.

Contrasting roles of condensin I and condensin II in mitotic chromosome formation.

In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-I-depleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.

Active transcription and essential role of RNA polymerase II at the centromere during mitosis.

Transcription of the centromeric regions has been reported to occur in G1 and S phase in different species. Here, we investigate whether transcription also occurs and plays a functional role at the mammalian centromere during mitosis. We show the presence of actively transcribing RNA polymerase II (RNAPII) and its associated transcription factors, coupled with the production of centromere satellite transcripts at the mitotic kinetochore. Specific inhibition of RNAPII activity during mitosis leads to a decrease in centromeric α-satellite transcription and a concomitant increase in anaphase-lagging cells, with the lagging chromosomes showing reduced centromere protein C binding. These findings demonstrate an essential role of RNAPII in the transcription of α-satellite DNA, binding of centromere protein C, and the proper functioning of the mitotic kinetochore.

Putative CENP-B paralogues are not present at mammalian centromeres.

Although centromere protein B (CENP-B) is a highly conserved mammalian centromere protein, its function remains unknown. The presence of the protein is required to form artificial satellite DNA-based centromeres de novo, yet cenpb knockout mice are viable for multiple generations with no mitotic or meiotic defects, and the protein is not present at fully functional neocentromeres. Previous studies have suggested that the presence of functionally redundant paralogues of CENP-B may explain the lack of a phenotype in knockout mice, and the related Tigger-derived (TIGD) family of proteins has been implicated as the most likely candidate for such paralogues. Here, we describe an investigation of the centromere-binding properties of the three TIGD proteins most highly related to CENP-B through phylogenetic analysis through EGFP fusion studies and immunocytochemistry. Although two of the three proteins bound to human centromeres with low affinity when overexpressed as fusion proteins, the strongest candidate, TIGD3, demonstrated no native centromeric binding when using raised antibodies, either in human cells or in cenpb (-/-) mouse ES cells. We conclude that the existence of functional CENP-B paralogues is highly unlikely and that CENP-B acts alone at the centromere. Based on these data, we suggest a new, meiotic drive model of CENP-B action during centromere repositioning in evolution.

Shorter telomere length in peripheral blood cells associated with migraine in women.

OBJECTIVE: To evaluate relative telomere length of female migraine patients. BACKGROUND: Migraine is a debilitating disorder affecting 6-28% of the population. Studies on the mechanisms of migraine have demonstrated genetic causes but the pathophysiology and subcellular effects of the disease remain poorly understood. Shortened telomere length is associated with age-related or chronic diseases, and induced stresses. Migraine attacks may impart significant stress on cellular function, thus this study investigates a correlation between shortening of telomeres and migraine. METHODS: Relative telomere length was measured using a previously described quantitative polymerase chain reaction method. A regression analysis was performed to assess differences in mean relative telomere length between migraine patients and healthy controls. RESULTS: The leukocyte telomeres of a cohort of 142 Caucasian female migraine subjects aged 18-77 years and 143 matched 17-77-year-old healthy control Caucasian women were examined. A significantly shorter relative telomere length was observed in the migraine group compared with the control group after adjusting for age and body mass index (P = .001). In addition, age of onset was observed to associate with the loss of relative telomere length, especially at early age of onset (<17 years old). No association was observed between relative telomere length and the severity and frequency of migraine attacks and the duration of migraine. CONCLUSION: Telomeres are shorter in migraine patients and there is more variation in telomere length in migraine patients.

ATRX interacts with H3.3 in maintaining telomere structural integrity in pluripotent embryonic stem cells.

ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. We demonstrate ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, we found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. We show by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomere-dysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin.

Rapid evolution of mouse Y centromere repeat DNA belies recent sequence stability.

The Y centromere sequence of house mouse, Mus musculus, remains unknown despite our otherwise significant knowledge of the genome sequence of this important mammalian model organism. Here, we report the complete molecular characterization of the C57BL/6J chromosome Y centromere, which comprises a highly diverged minor satellite-like sequence (designated Ymin) with higher-order repeat (HOR) sequence organization previously undescribed at mouse centromeres. The Ymin array is approximately 90 kb in length and resides within a single BAC clone that provides sequence information spanning an endogenous animal centromere for the first time. By exploiting direct patrilineal inheritance of the Y chromosome, we demonstrate stability of the Y centromere DNA structure spanning at least 175 inbred generations to beyond the time of domestication of the East Asian M.m. molossinus "fancy" mouse through which the Y chromosome was first introduced into the classical inbred laboratory mouse strains. Despite this stability, at least three unequal genetic exchange events have altered Ymin HOR unit length and sequence structure since divergence of the ancestral Mus musculus subspecies around 900,000 yr ago, with major turnover of the HOR arrays driving rapid divergence of sequence and higher-order structure at the mouse Y centromere. A comparative sequence analysis between the human and chimpanzee centromeres indicates a similar rapid divergence of the primate Y centromere. Our data point to a unique DNA sequence and organizational architecture for the mouse Y centromere that has evolved independently of all other mouse centromeres.

Orchestrating twosome and foursome chromosome parties.

The conserved centromere protein C (CENP-C) is indispensable for kinetochore function. Yet its mechanism of action has remained elusive. In this issue of Developmental Cell, Tanaka et al. report that the fission yeast homolog, Cnp3, acts as a linker protein that fulfills a variety of different roles in the bi- and mono-orientation of chromosomes during mitosis and meiosis I.

Improved methodology for assessment of mRNA levels in blood of patients with FMR1 related disorders.

BACKGROUND: Elevated levels of FMR1 mRNA in blood have been implicated in RNA toxicity associated with a number of clinical conditions. Due to the extensive inter-sample variation in the time lapse between the blood collection and RNA extraction in clinical practice, the resulting variation in mRNA quality significantly confounds mRNA analysis by real-time PCR. METHODS: Here, we developed an improved method to normalize for mRNA degradation in a sample set with large variation in rRNA quality, without sample omission. Initially, RNA samples were artificially degraded, and analyzed using capillary electrophoresis and real-time PCR standard curve method, with the aim of defining the best predictors of total RNA and mRNA degradation. RESULTS: We found that: (i) the 28S:18S ratio and RNA quality indicator (RQI) were good predictors of severe total RNA degradation, however, the greatest changes in the quantity of different mRNAs (FMR1, DNMT1, GUS, B2M and GAPDH) occurred during the early to moderate stages of degradation; (ii) chromatographic features for the 18S, 28S and the inter-peak region were the most reliable predictors of total RNA degradation, however their use for target gene normalization was inferior to internal control genes, of which GUS was the most appropriate. Using GUS for normalization, we examined in the whole blood the relationship between the FMR1 mRNA and CGG expansion in a non-coding portion of this gene, in a sample set (n = 30) with the large variation in rRNA quality. By combining FMR1 3' and 5' mRNA analyses the confounding impact of mRNA degradation on the correlation between FMR1 expression and CGG size was minimized, and the biological significance increased from p = 0.046 for the 5' FMR1 assay, to p = 0.018 for the combined FMR1 3' and 5' mRNA analysis. CONCLUSION: Our observations demonstrate that, through the use of an appropriate internal control and the direct analysis of multiple sites of target mRNA, samples that do not conform to the conventional rRNA criteria can still be utilized to obtain biologically/clinically relevant data. Although, this strategy clearly has application for improved assessment of FMR1 mRNA toxicity in blood, it may also have more general implications for gene expression studies in fresh and archival tissues.

Histone H3.3 incorporation provides a unique and functionally essential telomeric chromatin in embryonic stem cells.

Little is known about the telomere chromatin dynamics of embryonic stem (ES) cell. Here, we demonstrate localization of histone H3.3 at interphase telomeres and enrichment of Ser31-phosphorylated H3.3 at metaphase telomeres in pluripotent mouse ES cells. Upon differentiation, telomeric H3.3S31P signal decreases, accompanied by increased association of heterochromatin repressive marks and decreased micrococcal nuclease sensitivity at the telomeres. H3.3 is recruited to the telomeres at late S/G2 phase, coinciding with telomere replication and processing. RNAi-depletion of H3.3 induces telomere-dysfunction phenotype, providing evidence for a role of H3.3 in the regulation of telomere chromatin integrity in ES cells. The distinctive changes in H3.3 distribution suggests the existence of a unique and functionally essential telomere chromatin in ES cells that undergoes dynamic differentiation-dependent remodeling during the process of differentiation.

LINE retrotransposon RNA is an essential structural and functional epigenetic component of a core neocentromeric chromatin.

We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10) containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A-binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A-binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s) across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A-binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A-associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.

Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin.

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509-516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85-93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.

Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution.

Since the discovery of the first human neocentromere in 1993, these spontaneous, ectopic centromeres have been shown to be an astonishing example of epigenetic change within the genome. Recent research has focused on the role of neocentromeres in evolution and speciation, as well as in disease development and the understanding of the organization and epigenetic maintenance of the centromere. Here, we review recent progress in these areas of research and the significant insights gained.

Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere.

The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric alpha-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires alpha-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following alpha-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds alpha-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that alpha-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis.

Mouse telocentric sequences reveal a high rate of homogenization and possible role in Robertsonian translocation.

The telomere and centromere are two specialized structures of eukaryotic chromosomes that are essential for chromosome stability and segregation. These structures are usually characterized by large tracts of tandemly repeated DNA. In mouse, the two structures are often located in close proximity to form telocentric chromosomes. To date, no detailed sequence information is available across the mouse telocentric regions. The antagonistic mechanisms for the stable maintenance of the mouse telocentric karyotype and the occurrence of whole-arm Robertsonian translocations remain enigmatic. We have identified large-insert fosmid clones that span the telomere and centromere of several mouse chromosome ends. Sequence analysis shows that the distance between the telomeric T2AG3 and centromeric minor satellite repeats range from 1.8 to 11 kb. The telocentric regions of different mouse chromosomes comprise a contiguous linear order of T2AG3 repeats, a highly conserved truncated long interspersed nucleotide element 1 repeat, and varying amounts of a recently discovered telocentric tandem repeat that shares considerable identity with, and is inverted relative to, the centromeric minor satellite DNA. The telocentric domain as a whole exhibits the same polarity and a high sequence identity of >99% between nonhomologous chromosomes. This organization reflects a mechanism of frequent recombinational exchange between nonhomologous chromosomes that should promote the stable evolutionary maintenance of a telocentric karyotype. It also provides a possible mechanism for occasional inverted mispairing and recombination between the oppositely oriented TLC and minor satellite repeats to result in Robertsonian translocations.