High-resolution organization of mouse telomeric and pericentromeric DNA

We studied the organization of telomeric, major and minor satellite DNA sequences located in the pericentromeric regions of mouse telocentric and Robertsonian metacentric chromosomes by high-resolution fluorescence in situ hybridization. Molecular data have already proved that in telocentrics, from the physical chromosome end, telomeric sequences are followed by minor and then by major satellite DNA. We showed that the three families of repetitive DNA are organized as uninterrupted long-range cluster repeats and that there is no intermingling between telomeric and minor satellite DNA or between the major and the minor tandem repeats or with non-satellite DNA. The pericentromeric region of metacentric chromosomes consists of a small block of minor satellite DNA sandwiched between two blocks of major satellite DNA.     

Interplay between genome organization and epigenomic alterations of pericentromeric DNA in cancer

In eukaryotic genome biology, the genomic organization inside the three-dimensional(3 D) nucleus is highly complex, and whether this organization governs gene expression is poorly understood. Nuclear lamina(NL)is a filamentous meshwork of proteins present at the lining of inner nuclear membrane that serves as an anchoring platform for genome organization. Large chromatin domains termed as lamina-associated domains(LADs), play a major role in silencing genes at the nuclear periphery. The interaction of the NL and genome is dynamic and stochastic. Furthermore, many genes change their positions during developmental processes or under disease conditions such as cancer, to activate certain sorts of genes and/or silence others. Pericentromeric heterochromatin(PCH) is mostly in the silenced region within the genome, which localizes at the nuclear periphery. Studies show that several genes located at the PCH are aberrantly expressed in cancer. The interesting question is that despite being localized in the pericentromeric region,how these genes still manage to overcome pericentromeric repression. Although epigenetic mechanisms control the expression of the pericentromeric region, recent studies about genome organization and genome-nuclear lamina interaction have shed light on a new aspect of pericentromeric gene regulation through a complex and coordinated interplay between epigenomic remodeling and genomic organization in cancer.     

High frequency DNA rearrangements associated with mouse centromeric satellite DNA

A DNA transformed mouse cell line, generated by the microinjection of a pBR322 plasmid containing the herpes thymidine kinase (tk) gene, was observed to exhibit a high frequency of DNA rearrangement at the site of exogenous DNA integration. The instability in this cell line does not appear to be mediated by the tk inserts or the immediately adjacent mouse DNA, but instead may be a consequence of the larger host environment at the chromosomal site of tk insertion. Results obtained from restriction analysis, in situ chromosome hybridizations, and cesium chloride density-gradient fractionations indicate that the tk inserts are organized as a single cluster of direct and inverted repeats embedded within pericentromeric satellite DNA. To determine the molecular identity of the flanking host sequences, one of the mouse-tk junction fragments was cloned, and subsequent restriction and sequence analyses revealed that this DNA fragment consists almost entirely of classical mouse satellite DNA. On the basis of these observations, we suggest that the instability in this cell line may reflect the endogenous instability or fluidity of satellite DNA.     

Localization of a mouse centromeric DNA repeat in interphase nuclei

The position of a mouse DNA repeat located near the centromere of mouse chromosomes X, 11, 13, and 17 was examined in interphase nuclei of bone marrow and fibroblast cells by in situ hybridization of 3H- or biotin-labeled DNA probe 70-38. In most laboratory mouse strains this probe recognizes a single repeat cluster (DXWas70) close to the centromere of the mouse X chromosome. In a few mouse strains, a second locus (D11Was70, D13Was70, or D17Was70, depending on the mouse strain) is located near the centromere of an autosome. In interphase nuclei from mouse strains with the X-linked locus only, two distinct sites of hybridization were found in female mice and one in male mice. These two sites remained separated during the different phases of the cell cycle (G1, early S, late S, and G2) as demonstrated by in situ hybridization of the probe to flow-sorted nuclei. In interphase nuclei from mouse strains with both the X-linked locus and an autosomal locus, four distinct sites of hybridization were found in female mice and three in male mice. Further analysis of loci DXWas70 and D17Was70 showed that these loci were often located in the outer region of nuclei from bone marrow and fibroblast cells.     

Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes.

A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon. However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.     

Tet1 regulates epigenetic remodeling of the pericentromeric heterochromatin and chromocenter organization in DNA hypomethylated cells

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300-400 microm. The length of a given satellite II array usually reached 200 microm, corresponding to 2 x 10(3) kb of DNA in a given centromere.     

Strong epigenetic similarity between maize centromeric and pericentromeric regions at the level of small RNAs, DNA methylation and H3 chromatin modifications

Both kinetochore function and sister chromatid cohesion can depend upon pericentromere chromatin structure, and factors associated with heterochromatin have been proposed to have general, conserved roles in distinguishing centromeres and pericentromeres and in conferring pericentromere-intrinsic functions. We applied genome-wide sequencing approaches to quantify RNA expression, DNA methylation and histone modification distributions in maize (Zea mays), focusing on two maize chromosomes with nearly fully sequenced centromeres and pericentromeres. Aside from the presence of the Histone H3 variant common to all centromeres, Centromeric Histone H3 (CENH3), we found no RNA expression or chromatin modifications that clearly differentiate pericentromeres from either centromeres or from chromosome arms, nor did we identify an epigenetic signature that accurately predicts CENH3 location. RNA expression and chromatin modification frequencies were broadly associated with distance from centromeres, gradually peaking or dipping toward arms. When interpreted in the context of experimental data from other systems, our results suggest that centromeres may confer essential functions (such as cohesion retention) to flanking sequence regardless of the local heterochromatin profile.     

Conserved organization of centromeric chromatin in flies and humans

Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. We show that Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID is depleted by RNAi, it is replaced by H3, demonstrating flexibility of centromeric chromatin organization. Finally, contrary to models proposing that H3 and CID/CENP-A nucleosomes are replicated at different times in S phase, we show that interspersed H3 and CID/CENP-A chromatin are replicated concurrently during S phase in humans and flies. We propose that the unique structural arrangement of CID/CENP-A and H3 nucleosomes presents centromeric chromatin to the poleward face of the condensing mitotic chromosome.     

How to build a centromere: from centromeric and pericentromeric chromatin to kinetochore assembly.

The assembly of the centromere, a specialized region of DNA along with a constitutive protein complex which resides at the primary constriction and is the site of kinetochore formation, has been puzzling biologists for many years. Recent advances in the fields of chromatin, microscopy, and proteomics have shed a new light on this complex and essential process. Here we review recently discovered mechanisms and proteins involved in determining mammalian centromere location and assembly. The centromeric core protein CENP-A, a histone H3 variant, is hypothesized to designate centromere localization by incorporation into centromere-specific nucleosomes and is essential for the formation of a functional kinetochore. It has been found that centromere localization of centromere protein A (CENP-A), and therefore centromere determination, requires proteins involved in histone deacetylation, as well as base excision DNA repair pathways and proteolysis. In addition to the incorporation of CENP-A at the centromere, the formation of heterochromatin through histone methylation and RNA interference is also crucial for centromere formation. The assembly of the centromere and kinetochore is complex and interdependent, involving epigenetics and hierarchical protein-protein interactions.     

Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.     

Nucleosome organization on Kluyveromyces lactis centromeric DNAs

The preferential assembly of specialized nucleosomes on budding yeast centromeres can be due either to the higher stability of specialized centromeric nucleosomes and/or to the lower stability of canonical centromeric nucleosomes with respect to bulk nucleosomes. We have evaluated the thermodynamic stability of canonical nucleosomes, assembled on Kluyveromyces lactis centromeric DNAs, with a competitive reconstitution assay and a theoretical method recently developed by us. The results, obtained by both methods, show that all five known centromeric DNAs from K. lactis are able to organize canonical nucleosomes, characterized by higher stability with respect those of bulk DNA. With 'footprinting' and theoretical prediction, based on sequence-dependent DNA elasticity, we have found that centromeric canonical nucleosomes are characterized by nucleosome dyad axis multiple positioning, rotationally phased. The isoenergetic nucleosome multiple positions are relevant in understanding the transition from canonical to specialized nucleosomes in interacting with centromere protein complexes. The satisfactory agreement between the results obtained from theoretical and experimental methods shows that sequence-dependent centromeric DNA elasticity has a main role in nucleosome thermodynamic stability and positioning.     

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere. Edited by: H. F. Willard     

The organization of the mouse satellite DNA at centromeres

The mouse genome contains a major and a minor satellite DNA family of repetitive DNA sequences. The use of 5-azacytidine has allowed us to demonstrate that these satellite DNAs are organized in two separate domains at the centromeres of mouse chromosomes. The minor satellite is closer to the short arms of the acrocentric chromosomes than the major satellite. The major satellite is farther away, flanking the minor satellite and adjacent to the euchromatic long arm of each mouse chromosome. At the level of resolution afforded by the in situ hybridization technique it would appear that the organization of the centromeric domain of the mouse is similar to that in man. That is, both contain two repetitive DNA sequence families arranged in major blocks.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag⁺-Cs₂SO₄ density gradient centrifugation. — According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2×10⁴ copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).— Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides. —Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides. — The organization of mouse genome analyzed by Ag⁺-Cs₂SO₄ density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Histone variants in mouse centromeric chromatin

Summary Highly purified centromeric heterochromatin was isolated from mouse liver nuclei and the pattern of core histone variants was analyzed. In comparison with total chromatin, the centromeric heterochromatin of young animals was characterized by (1) enrichment in the replication-dependent variants H2A₁, H2B₂ and H3₂, (2) reduced amount of the minor variant H2A_z and (3) absence of ubiquitinated molecules of H2A. This specific variant pattern changed upon ageing as a result of accumulation of replacement variants so that in adult animals both chromatin preparations exhibited similar pattern for H2A and H2B, while the difference in the profile of H3 variants was preserved.     

Molecular organization and haplotype analysis of centromeric DNA from human chromosome 17: Implications for linkage in neurofibromatosis

The alpha satellite DNA subset located at the centromere of human chromosome 17 has been shown to be tightly linked genetically to the gene for von Recklinghausen neurofibromatosis (NF1). The centromeric DNA polymorphisms used for linkage analyses in NF1 are complex and involve a "locus" (D17Z1) that spans over one million base pairs of satellite DNA. To understand more completely the basis for these polymorphisms and how they might be best scored and used in the analysis of NF1, we have examined the molecular composition of the alpha satellite array on individual copies of chromosome 17 by two complementary approaches. First, we have analyzed segregation of chromosome 17 alpha satellite haplotypes in large, three-generation families that provide information on the different types of alpha satellite segregating in a block fashion. Second, we have analyzed directly the extent of variation in different D17Z1 arrays by genomic blotting analysis of haploid copies of chromosome 17 isolated in rodent/human somatic cell hybrids. The data indicate the existence of a wide range of different alpha satellite variants on individual copies of chromosome 17, each haplotype differing in the size, restriction map, and relative proportion of particular polymorphic repeat forms. Despite this complexity, the D17Z1 markers provide a potentially useful and genetically close starting point for the molecular and clinical analysis of NF1.