Centromeric Elements Research Articles

Chromosome-Level Assemblies for the Pine Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome's distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen.

Comparative Analysis of Transposable Elements and the Identification of Candidate Centromeric Elements in the Prunus Subgenus Cerasus and Its Relatives.

The subgenus Cerasus and its relatives include many crucial economic drupe fruits and ornamental plants. Repetitive elements make up a large part of complex genomes, and some of them play an important role in gene regulation that can affect phenotypic variation. However, the variation in their genomes remains poorly understood. This work conducted a comprehensive repetitive sequence identification across the draft genomes of eight taxa of the genus Prunus, including four of the Prunus subgenus Cerasus (Prunus pseudocerasus, P. avium, P. yedoensis and P. × yedoensis) as well as congeneric species (Prunus salicina, P. armeniaca, P. dulcis and P. persica). Annotation results showed high proportions of transposable elements in their genomes, ranging from 52.28% (P. armeniaca) to 61.86% (P. pseudocerasus). The most notable differences in the contents of long terminal repeat retrotransposons (LTR-RTs) and tandem repeats (TRs) were confirmed with de novo identification based on the structure of each genome, which significantly contributed to their genome size variation, especially in P. avium and P. salicina. Sequence comparisons showed many similar LTR-RTs closely related to their phylogenetic relationships, and a highly similar monomer unit of the TR sequence was conserved among species. Additionally, the predicted centromere-associated sequence was located in centromeric regions with FISH in the 12 taxa of Prunus. It presented significantly different signal intensities, even within the diverse interindividual phenotypes for Prunus tomentosa. This study provides insight into the LTR-RT and TR variation within Prunus and increases our knowledge about its role in genome evolution.

Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering.

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

Defining the consequences of endogenous genetic variation within a novel family of Schizosaccharomyces pombe heterochromatin nucleating sequences.

Centromeres are essential for genetic inheritance—they prevent aneuploidy by providing a physical link between DNA and chromosome segregation machinery. In many organisms, centromeres form at sites of repetitive DNAs that help establish the chromatin architecture required for centromere function. These repeats are often rapidly evolving and subject to homogenization, which causes the expansion of novel repeats and sequence turnover. Thus, centromere sequence varies between individuals and across species. This variation can affect centromere function. We utilized Schizosaccharomyces pombe to assess the relationship between centromere sequence and chromatin structure and determine how sensitive this relationship is to genetic variation. In S. pombe, nucleating sequences within centromere repeats recruit heterochromatin via multiple mechanisms, which include RNA-interference (RNAi) . Heterochromatin, in turn, contributes to centromere function through its participation in three essential processes; establishment of a kinetochore, cohesion of sister chromatids, and suppression of recombination. Here, we show that a centromere element containing RevCen, a target of the RNAi pathway, establishes heterochromatin and gene silencing when relocated to a chromosome arm. Within this RevCen-containing element (RCE), a highly conserved domain is necessary for full heterochromatin nucleation but cannot establish heterochromatin independently. We characterize the 10 unique RCEs in the S. pombe centromere assembly, which range from 60% to 99.6% identical, and show that all are sufficient to establish heterochromatin. These data affirm the importance of centromere repeats in establishing heterochromatin and suggest there is flexibility within the sequences that mediate this process. Such flexibility may preserve centromere function despite the rapid evolution of centromere repeats.

Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses

BackgroundCultivated grasses are an important source of food for domestic animals worldwide. Increased knowledge of their genomes can speed up the development of new cultivars with better quality and greater resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass species (Lolium) and diploid and hexaploid fescue species (Festuca). In this work, we characterized repetitive DNA sequences and their contribution to genome size in five fescue and two ryegrass species as well as one fescue and two ryegrass cultivars.ResultsPartial genome sequences produced by Illumina sequencing technology were used for genome-wide comparative analyses with the RepeatExplorer pipeline. Retrotransposons were the most abundant repeat type in all seven grass species. The Athila element of the Ty3/gypsy family showed the most striking differences in copy number between fescues and ryegrasses. The sequence data enabled the assembly of the long terminal repeat (LTR) element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of fluorescence in situ hybridization (FISH) with a probe specific to the Fesreba element and immunostaining with centromeric histone H3 (CENH3) antibody showed their co-localization and indicated a possible role of Fesreba in centromere function.ConclusionsComparative repeatome analyses in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of the LTR element Fesreba. A new LTR element Fesreba was identified and found in abundance in centromeric regions of the fescues and ryegrasses. It may play a role in the function of their centromeres.

Dgcr8 is Indispensable for Cardiac Lineage Specification in Embryonic Stem Cells

Objective: microRNAs have been shown to play important roles in cellular behavior and lineage specification including cardiogenic differentiation. However, full understanding of their roles in cardiomyocyte differentiation has been impeded due to lack of proper cellular model. Here, we used an embryonic stem cell (ESC) that is lacking the important microprocessor Dgcr8 (or Pasha), which allows the introduction of individual miRNAs to study their role in cardiac differentiation and for more precise target selection. Methods: Dgcr8 KO ESC was cultured in LIF-supplemented ESC medium with mouse embryonic fibroblast feeders and cardiac differentiation was induced using an embryonic body-based differentiation protocol. Differentiation was monitored by measuring mRNA and protein levels of cardiogenic markers and heterochromatin changes using immunofluorescent staining and semi-quantitative PCR. Results and conclusion: We showed that Dgcr8 KO ESCs indeed are lacking a large population of small RNAs, including but not limited to mature microRNAs. The KO cells had a lower proliferation rate and were unable to differentiate into the cardiac lineage. To our surprise, in addition to a defect in microRNA processing, Dgcr8 KO embryonic stem cells are unable to form proper heterochromatin and to inactivate genotoxic centromeric repetitive elements. Our results argue that, in addition to controlling microRNA processing, Dgcr8 may serve a previously unrecognized role in heterochromatin silencing.

22 OXAMFLATIN TREATMENT ENHANCES NUCLEAR REPROGRAMMING BY INHIBITING XIST EXPRESSION AND REDUCING DNA METHYLATION IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) enhances developmental potential by alteration of epigenetic status. Oxamflatin is one of the potent HDACi. In our previous study, development to Day 7 blastocysts was enhanced when the porcine somatic cell nuclear transfer (SCNT) embryos were treated with oxamflatin for 16 h. The objective of the present study was to investigate the effect of oxamflatin treatment on XIST gene expression and DNA methylation of XIST gene and centromeric repeat element in Day 7 SCNT blastocysts. Somatic cell nuclear transfer was performed on enucleated metaphase II oocytes using a transgene female cell line. Cloned embryos were electrically fused and activated, treated with 150 nM oxamflatin for 16 h and cultured in PZM3 under 5% CO2, 5% oxygen, and 90% N2 for 7 days. Clones without Oxamflatin treatment were used as controls. For XIST methylation, IVF blastocysts at Day 7 were used as controls. Blastocysts at Day 7 were pooled from each treatment group and processed for methylation analysis by bisulfite sequencing and gene expression by quantitative real-time PCR. This experiment was replicated 4 times. The percent of CpG methylation in donor cells before SCNT was also determined. Data were analysed by using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). In donor cells, 45.3 ± 5.8% of CpGs in a centromeric repeat element (9 CpGs in GenBank Z75640) were methylated. In the SCNT embryos, oxamflatin treatment reduced methylation from 27.3 ± 3.1% in the control to 18.2 ± 3.2% (P < 0.05). The average methylation in XIST (11 CpGs in GenBank KC149530.1) in donor cells was 42.4 ± 6.4%. This CpG island had 2 sites that were not methylated in any of the samples. However, the remaining 9 CpGs were methylated in 8 of 15 samples; for example, showing a parental imprint of ~50%. This implied that the CpG island studied represented the real-time status of the XIST locus in the cell and provides a good marker for reprogramming studies. XIST methylation level in Day 7 blastocysts was not different between oxamflatin (11.8 ± 3.2%) and control (11.8 ± 3.2%). However, XIST methylation in SCNT embryos was higher than in the same age IVF blastocysts (11.7 ± 1.7 v. 0.6 ± 2.4%; P < 0.01). Oxamflatin treatment tended to decrease XIST expression in Day 7 blastocysts compared with controls (18.8 ± 0.8 v. 21.7 ± 0.8; P < 0.1) as measured by real-time PCR. Interestingly, XIST gene expression was positively correlated with its methylation (P < 0.05). In conclusion, these results indicate that during nuclear reprogramming there was a dramatic decrease in DNA methylation from donor cells to Day 7 SCNT embryos. The higher methylation of XIST in SCNT embryos compared with IVF embryos suggests that the reprogramming of donor cells was not completed, which may be a contributor to low cloning efficiency. Oxamflatin treatment of SCNT embryos may enhance nuclear reprogramming by inhibiting XIST expression and reducing DNA methylation, resulting in better embryo development.

HULC, a Histone H2B Ubiquitinating Complex, Modulates Heterochromatin Independent of Histone Methylation in Fission Yeast

Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures.

A hemicentric inversion in the maize line knobless Tama flint created two sites of centromeric elements and moved the kinetochore-forming region

A maize line, knobless Tama flint (KTF), was found to contain a version of chromosome 8 with two spatially distinct regions of centromeric elements, one at the original genetic position and the other at a novel location on the long arm. The new site of centromeric elements functions as the kinetochore-forming region resulting in a change of arm length ratio. Examination of fluorescence in situ hybridization markers on chromosome 8 revealed an inversion between the two centromere sites relative to standard maize lines, indicating that this chromosome 8 resulted from a hemicentric inversion with one breakpoint approximately 20 centi-McClintocks (cMc) on the long arm (20% of the total arm length from the centromere) and the other in the original cluster of centromere repeats. This inversion moved the kinetochore-forming region but left the remainder of the centromere repeats. In a hybrid between a standard line (Mo17) and KTF, both chromosome 8 homologues were completely synapsed at pachytene despite the inversion. Although the homologous centromeres were not paired, they were always correctly oriented at anaphase and migrated to opposite poles. Additionally, recombination on 8L was severely repressed in the hybrid.

Karyotypic evolution and organization of the highly repetitive DNA sequences in the Japanese shrew-moles, Dymecodon pilirostris and Urotrichus talpoides

The karyological relationship and organization of highly repetitive DNA sequences in Japanese shrew-moles were studied by zoo-blot hybridization and fluorescence in situ hybridization (FISH). When the genomic DNA of the eastern race of Urotrichus talpoides was digested with PstI, three fragments of highly repetitive DNA sequences, approximately 0.7, 0.9, and 1.4 kb in length, were observed as distinct bands. The results of FISH in the eastern race of U. talpoides using these three fragments separately as probes showed that the 0.7-kb PstI fragment was distributed in the centromeric regions of most chromosomes, and that the 0.9- and 1.4-kb fragments were predominantly located in the C-heterochromatin region of chromosome 13p. Although the western race of U. talpoides also had three PstI fragments, 0.9- and 1.4-kb PstI fragments were more ambiguous than those of the eastern race. The PstI- digested genomic DNA in Dymecodonpilirostris produced only a faint 0.9-kb band, and its signal patterns obtained by zoo-blot hybridization were clearly different from those of U. talpoides. The 0.7-kb fragment of U. talpoides hybridized strongly with the 0.9-kb fragment of D. pilirostris. In a FISH analysis, the 0.9-kb fragment of D. pilirostris hybridized with highly repetitive DNA in the centromeric regions of most chromosomes from both D. pilirostris and U. talpoides. Zoo-blot hybridization and FISH analyses suggest that the 0.9- and 1.4-kb PstI fragments were generated specifically in the genome of U. talpoides after the common ancestor differentiated into two extant shrew-mole species. A difference in the length of the centromeric elements between U. talpoides and D. pilirostris might be observed due to certain modifications of the repeating unit.

Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein.

The papillomavirus E2 protein functions in viral transcriptional regulation, DNA replication, and episomal genome maintenance. Viral genomes are maintained in dividing cells by attachment to mitotic chromosomes by means of the E2 protein. To investigate the chromosomal tethering function of E2, plasmid stability assays were developed in Saccharomyces cerevisiae to determine whether the E2 protein could maintain plasmids containing the yeast autonomous replication sequence replication element but with the centromeric element replaced by E2-binding sites. E2 expression was not sufficient to maintain such plasmids, but plasmid stability could be rescued by expression of the mammalian protein Brd4. In the presence of both Brd4 and E2 proteins, plasmids with multiple E2-binding sites were stable without selection. S. cerevisiae encodes a homolog of Brd4 named Bdf1 that does not contain the C-terminal domain that interacts with the E2 protein. A fusion protein of Bdf1 and the Brd4 C-terminal "tail" could support E2-mediated plasmid maintenance in yeast. Using a panel of mutated E2 proteins, we determined that plasmid stability required the ability of E2 to bind DNA and to interact with Brd4 and mammalian mitotic chromosomes but did not require its replication initiation and transactivation functions. The S. cerevisiae-based plasmid maintenance assays described here are invaluable tools for dissecting mechanisms of episomal viral genome replication and screening for additional host protein factors involved in plasmid maintenance.

Analysis of Mitotic and Expression Properties of Human Neocentromere-based Transchromosomes in Mice

Human neocentromeres are functional centromeres that are devoid of the typical human centromeric alpha-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model.

Genetic interactions between an RNA polymerase II phosphatase and centromeric elements in Saccharomyces cerevisiae

The Saccharomyces cerevisiase protein phosphatase Fcp1 has been implicated in the regulation of transcription by RNA polymerase II, and is encoded by the essential gene FCP1. A screen was carried out for multicopy suppressors of the temperature-sensitive phenotype of two phosphatase mutants, fcp1-2 and fcp1-4. Only the wild-type FCP1 was found to suppress (complement) the fcp1-4 mutation. For fcp1-2 three second-site suppressors were identified. One contained the ORF for ZDS1. The remaining two suppressors mapped to the centromere regions of chromosomes I and V. Suppression due to centromere DNA was found to be more dependent on the CDEIII region than on other regions of the centromere. The presence of a suppressor centromere affected the level of Fcp1 protein and the overall phosphorylation state of RNA polymerase II (RNAPII) in fcp1-2 cells, but not wild-type cells, grown at both permissive and non-permissive temperatures. In addition, genetic interactions were identified between this FCP1 mutant and the genes SKP1, CEP3 and CBF1, which code for centromere binding proteins. The mechanism of suppression and regulation of Fcp1-2 protein activity by centromeric DNA is discussed.

Plasmid Accumulation Reduces Life Span in Saccharomyces cerevisiae

Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction. Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth. The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.

Minichromosome formation in Chlorella cells irradiated with electron beams

Three minichromosomes, miniP7, miniB7, and miniK4 of 800 kbp, 450 kbp, and 550 kbp, respectively, were obtained from Chlorella vulgaris chromosome I by electron-beam irradiation. Two of them were structurally characterized: MiniP7 was formed by the deletion of an internal 180 kbp close to the right end of chromosome I. The 180-kbp region with a small interspersed nuclear element (SINE)-like element on its left terminus was translocated to another chromosome, leaving a footprint-like structure on miniP7. MiniB7 was a hybrid of chromosome I and another chromosome, retaining the left telomere and the centromere of chromosome I. The centromeric repetitive elements served as a rearrangement point in the miniB7 formation. These examples showed the complicated mechanisms involved in the minichromosome formation. The minichromosomes thus obtained can be useful for isolating the fundamental structural elements of a chromosome. Moreover, they may serve as starting materials or a vector to generate artificial chromosomes carrying useful genes.

Cis-Acting DNA from Fission Yeast Centromeres Mediates Histone H3 Methylation and Recruitment of Silencing Factors and Cohesin to an Ectopic Site

Background: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established.Results: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9.Conclusions: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.

Centromeres can run, but they can’t hide (from kinetochores)

Centromeres are the specialized regions of eukaryotic chromatin responsible for the physical association of chromosomes with the mitotic spindle. In particular, a large proteinaceous structure known as the kinetochore assembles onto the centromeric regions of chromosomes and mediates their attachment to spindle microtubules. While it has long been appreciated that these attachments are essential for proper chromosome segregation during mitosis, the process by which kinetochore proteins associate exclusively with centromeres (as opposed to associating non-specifically with bulk chromatin) is not well understood.The simplest explanation for this association is that there exist ‘kinetochore-anchoring’ factors that bind to specific DNA sequences found exclusively at centromeres. For instance, in the budding yeast Saccharomyces cerevisiae, a unique ∼125-bp CEN sequence (derived from yeast centromere DNA) is sufficient to confer proper chromosome segregation activity to episomal DNA (e.g. plasmids). However, a similarly active centromeric sequence element has not been demonstrated in higher eukaryotes.Nonetheless, it has been demonstrated that there exist unique, highly repetitive sequences at the centromeres of various higher eukaryotes. For example, human centromeres are characterized by long (up to 5 Mb) repeats of a ∼171-bp sequence known as α-satellite DNA. These α-satellite sequences can associate with known kinetochore proteins in vitro and in vivo, but DNA fragments encoding α-repeats are not sufficient to confer spindle attachment reliably to artificial chromosomes. Thus, kinetochore assembly at centromeres could be an epigenetic phenomenon in higher metazoans.Consistent with this possibility, mutant chromosomes (presumably arising from mitotic recombination events) have been discovered in which kinetochore components assemble onto regions of chromatin that are not at the normal centromeric position. Most significantly, these ‘neocentromeres’ lack detectable α-satellite DNA sequences but allow microtubule attachment and confer proper chromosome segregation during mitosis.In this study, the authors used a combination of fluorescence in-situ hybridization (FISH) and immunofluorescence to detect cytologically the presence of 22 different kinetochore proteins on wild-type and neocentromere-containing human chromosomes1xHuman centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins. Saffery, R. et al. Hum. Mol. Genet. 2000; 9: 175–185Crossref | PubMedSee all

A simple signal element mediates transcription termination and mRNA 3' end formation in the DEG1 gene of Saccharomyces cerevisiae.

DEG1 is a weakly transcribed gene of Saccharomyces cerevisiae, closely associated with CEN6. We mapped its major poly(A) site only 24 nucleotides (nt) downstream of the stop codon, and only 26 nt upstream of the CDEI centromere element. The deletion of this 50 nt stretch completely abolishes formation of the mRNA 3' end. A shorter deletion of a 16 nt sequence in the 3'-untranslated region has the same effect on transcription termination and 3'-maturation function. A TATATA sequence within this 16 nt region is essential for both functions, while a TGTATA sequence has a weak compensating activity in 3' end maturation if the TATATA stretch is deleted. We assume that the 3' end formation signals of the DEG1 gene have this simple structure: a single essential element (TATATA, whether alone or with the few surrounding nucleotides), probably, but not necessarily, cooperating with the sequence at the poly(A) site. This simple structure differs from the emerging model for 3' end-processing signals in that (i) it is shorter: 24 nt long at the most, while the model suggests 39 nt; (ii) there is no element located downstream of the TATATA signal to position the poly(A) site; and (iii) unlike the other naturally occurring signals studied, no cooperation among multiple TATATA-like elements is observed. We found that the same TATATA sequence also directs transcription termination, irrespective of promoter strength, and presumably without the cooperation of a downstream polymerase II pausing site. Taken together, these findings support the hypothesis that the DEG1 3' end-forming signals are more condensed than in other yeast genes, probably because of their proximity to CEN6.

CDEBP, a site-specific DNA-binding protein of the 'APP-like' family, is required during the early development of the mouse.

A murine protein, termed CDEBP, was previously shown to bind the double-stranded DNA motif GTCACATG, identical to the yeast centromeric element CDEI. The cDNA sequence showed three domains with extensive similarities to the amyloid beta precursor protein (APP). The protein is homologous over its entire length to the human protein designated APPH. In situ immunofluorescence assays using antibodies raised against distinct parts of CDEBP detected discrete sites of accumulation inside the interphase nucleus, and the bulk of the protein was not associated with mitotic chromosomes. One of the complexes with double-stranded CDEI oligonucleotides detected by gel shift assay was not present when the protein had been selectively removed from nuclear extracts by immunoprecipitation. We reported previously that microinjection into one-cell mouse embryos of DNA fragments including the CDEI sequence results in an early arrest of development with abnormal nuclei containing variable amounts of DNA. The same characteristic figures were observed when embryos were treated with antisense oligonucleotides complementary to parts of the CDEBP coding region. Complexes between the CDEBP protein and CDEI sites in the mouse genome thus appear to play a critical role in the replication/segregation of the embryonic genome.

Identification and characterization of a spinal muscular atrophy-determining gene

Spinal muscular atrophy (SMA) is a common fatal autosomal recessive disorder characterized by degeneration of lower motor neurons, leading to progressive paralysis with muscular atrophy. The gene for SMA has been mapped to chromosome 5q13, where large-scale deletions have been reported. We describe here the inverted duplication of a 500 kb element in normal chromosomes and narrow the critical region to 140 kb within the telomeric region. This interval contains a 20 kb gene encoding a novel protein of 294 amino acids. An highly homologous gene is present in the centromeric element of 95% of controls. The telomeric gene is either lacking or interrupted in 226 of 229 patients, and patients retaining this gene (3 of 229) carry either a point mutation (Y272C) or short deletions in the consensus splice sites of introns 6 and 7. These data suggest that this gene, termed the survival motor neuron (SMN) gene, is an SMA-determining gene.