Chromatin conformation signatures of cellular differentiation

One of the major genomics challenges is to better understand how correct gene expression is orchestrated. Recent studies have shown how spatial chromatin organization is critical in the regulation of gene expression. Here, we developed a suite of computer programs to identify chromatin conformation signatures with 5C technology http://Dostielab.biochem.mcgill.ca. We identified dynamic HoxA cluster chromatin conformation signatures associated with cellular differentiation. Genome-wide chromatin conformation signature identification might uniquely identify disease-associated states and represent an entirely novel class of human disease biomarkers.     

Replication forks pause at yeast centromeres.

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.     

Time of replication of yeast centromeres and telomeres

The time of replication of centromeres and telomeres of the yeast S. cerevisiae was determined by performing Meselson-Stahl experiments with synchronized cells. The nine centromeres examined become hybrid in density early in S phase, eliminating the possibility that a delay in the replication of centromeres until mitosis is responsible for sister chromatid adherence and proper chromosome segregation at anaphase. The conserved sequence element Y', present at most telomeres, replicates late in S phase, as do the unique sequences adjacent to five specific telomeres. The early and late replication times of these structural elements may be either essential for their proper function or a consequence of some architectural feature of the chromosome.     

Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.     

Chromatin assembly in yeast cell-free extracts

A simple method for preparing chromatin assembly extracts has not been available for budding yeast. Here I describe such a method in detail. The assembly extract, a crude 100,000g supernatant, is prepared from cells disrupted in a manual or motorized grinder while they are frozen. The core histones and all soluble protein factors required for chromatin assembly under physiological conditions are present in the extract. Assembly is sensitive to mutation of lysine residues in the amino-terminal tail of histone H4 whose acetylation is associated with nucleosome deposition in vivo. The reaction is ATP dependent, and assembly-driven DNA supercoiling occurs with the same efficiency as in extracts from mammalian somatic cells. This simple system offers a unique opportunity to analyze chromatin metabolism by a combined biochemical and genetic approach that is not feasible for any other model organism.     

Rapid evolution of yeast centromeres in the absence of drive

To find the most rapidly evolving regions in the yeast genome we compared most of chromosome III from three closely related lineages of the wild yeast Saccharomyces paradoxus. Unexpectedly, the centromere appears to be the fastest-evolving part of the chromosome, evolving even faster than DNA sequences unlikely to be under selective constraint (i.e., synonymous sites after correcting for codon usage bias and remnant transposable elements). Centromeres on other chromosomes also show an elevated rate of nucleotide substitution. Rapid centromere evolution has also been reported for some plants and animals and has been attributed to selection for inclusion in the egg or the ovule at female meiosis. But Saccharomyces yeasts have symmetrical meioses with all four products surviving, thus providing no opportunity for meiotic drive. In addition, yeast centromeres show the high levels of polymorphism expected under a neutral model of molecular evolution. We suggest that yeast centromeres suffer an elevated rate of mutation relative to other chromosomal regions and they change through a process of "centromere drift," not drive.     

Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres

The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1).     

Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast

The spindle assembly checkpoint is the mechanism or set of mechanisms that prevents cells with defects in chromosome alignment or spindle assembly from passing through mitosis. We have investigated the effects of mini-chromosomes on this checkpoint in budding yeast by performing pedigree analysis. This method allowed us to observe the frequency and duration of cell cycle delays in individual cells. Short, centromeric linear mini-chromosomes, which have a low fidelity of segregation, cause frequent delays in mitosis. Their circular counterparts and longer linear mini-chromosomes, which segregate more efficiently, show a much lower frequency of mitotic delays, but these delays occur much more frequently in divisions where the mini-chromosome segregates to only one of the two daughter cells. Using a conditional centromere to increase the copy number of a circular mini-chromosome greatly increases the frequency of delayed divisions. In all cases the division delays are completely abolished by the mad mutants that inactivate the spindle assembly checkpoint, demonstrating that the Mad gene products are required to detect the subtle defects in chromosome behavior that have been observed to arrest higher eukaryotic cells in mitosis.     

Chromatin folding modulates nucleosome positioning in yeast minichromosomes

Based on the chromatin structures of the yeast URA3 gene and the TRP1ARS1 circle, we have designed circular minichromosomes of different sizes that should each form a tight tetranucleosome. This structure was assumed to be stiff and bulky and therefore likely to be sensitive to packaging into a three-dimensional structure. The structures of the minichromosomes were determined using micrococcal nuclease. Only one of the minichromosomes showed a protected region of about 570 bp, compatible with the predicted tight tetranucleosome, while all other constructs showed alternative structures. A comparison of the structures revealed that neither histone-DNA interactions nor influences from flanking boundaries are sufficient determinants of nucleosome positions. The data strongly suggest that chromatin folding modulates the nucleosome arrangement along the DNA.     

Subunit conformation of yeast alcohol dehydrogenase.

The primary structure of yeast alcohol dehydrogenase has been compared to the known tertiary structure of the corresponding horse liver enzyme after proper alignment of the two proteins. Possible influences on the subunit conformations of all amino acid exchanges, which affect 75% of the positions, were examined from interactions in the x-ray model of the horse enzyme. In spite of the differences, 90 of 93 strictly internal residues are similar, 18 space-restricted glycine residues are conserved, 16 structurally compensated exchanges occur, all functionally essential residues are similar or identical, and 41 gaps in either sequence may be accommodated in the model. These results show that the general subunit conformations and enzymatic mechanisms of the two enzymes are largely identical. Four surface areas are changed, affecting a region with differing charges, a noncommon loop, a structure around the second zinc atom, and residues at the main dimer interface. Although the subunit interactions in the yeast enzyme cannot be determined, the surface changes probably correlate with differences in quaternary structure between the proteins.     

The domain structure of centromeres is conserved from fission yeast to humans

The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.     

Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast

Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin of a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase II, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm1 mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.     

Chromatin conformation: a systematic analysis of helical parameters from hydrodynamic data

Chromatin has a “bead-and-bridge” appearance when viewed by electron microscopy. We have used quasielastic light scattering and sedimentation velocity techniques to study the hydrodynamic properties of chicken erythrocyte chromatin multimers in an attempt to determine the superstructure in solution. The functional dependence of the friction factor on the number of core particles in the multimer was analyzed by the Garcia de la Torre-Bloomfield formalism for a rigid array of odd-sized beads. The hydrodynamic parameters of the monomeric and dimeric subunit components, i.e., bead size and separation, form the basis of a systematic determination of the superstructure. These calculations support a helical conformation for chromatin multimers containing up to twenty repeat units. It is also shown that an “equivalent” helix can be obtained if the bead separation distance is not constrained to that determined for the dimer.