Comparison of the three-dimensional organization of sperm and fibroblast genomes using the Hi-C approach

BackgroundThe three-dimensional organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts.ResultsThe obtained data demonstrate that the three-dimensional genome organizations of sperm and fibroblast cells show a high degree of similarity both with each other and with the previously described mouse embryonic stem cells. Both A- and B-compartments and topologically associated domains are present in spermatozoa and fibroblasts. Nevertheless, sperm cells and fibroblasts exhibit statistically significant differences between each other in the contact probabilities of defined loci. Tight packaging of the sperm genome results in an enrichment of long-range contacts compared with the fibroblasts. However, only 30% of the differences in the number of contacts are based on differences in the densities of their genome packages; the main source of the differences is the gain or loss of contacts that are specific for defined genome regions. We find that the dependence of the contact probability on genomic distance for sperm is close to the dependence predicted for the fractal globular folding of chromatin.ConclusionsOverall, we can conclude that the three-dimensional structure of the genome is passed through generations without being dramatically changed in sperm cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0642-0) contains supplementary material, which is available to authorized users.     

The centromere of budding yeast

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in tethe cell cycle.     

Structural analysis of a yeast centromere

The most striking region of structural differentiation of a eukaryotic chromosome is the kinetochore. This chromosomal domain plays an integral role in the stability and propagation of genetic material to the progeny cells during cell division. The DNA component of this structure, which we refer to as the centromere, has been localized to a small region of 220–250 base pairs within the chromosomes from the yeast Saccharomyces cerevisiae. The centromere DNA (CEN) is organized in a unique structure in the cell nucleus and is required for chromosome stability during both mitotic and meiotic cell cycles. The centromeres from one chromosome can stabilize small circular minichromosomes or other yeast chromosomes. The centromeres may therefore interact with the same components of the segregation apparatus regardless of the chromosome in which they reside. The CEN DNA does not encode any regulatory RNAs or proteins, but rather is a cis-acting element that provides genetic stability to adjacent DNA sequences.     

Cytological identification of the genomes in pentaploidAllium neapolitanum using Giemsa C-banding

Two homoeologous sets of chromosomes in pentaploidAllium neapolitanum Cyr. (Liliaceae) are recognizable by their C-bands. The banding pattern is the same for one Californian and five Yugoslavian populations, suggesting a common chromosomal origin. Predominant meiotic association of identically banded homologues indicates a genomic formula of AA A_I BB, confirms an allopolyploid origin, and argues against genetically controlled pairing.     

UV-induced damage and repair in centromere DNA of yeast

Summary The centromere is the region within a chromosome that is required for proper segregation during mitosis and meiosis. Lesions in this sequence represent a unique type of damage, as loss of function could result in catastrophic loss of the genetic material of an entire chromosome. We have measured the induction by ultraviolet (UV) light of pyrimidine dimers in a 2550-bp restriction fragment that includes the centromere region of chromosome III in Saccharomyces cerevisiae. Yeast cells were exposed to ultraviolet light, cellular DNA was gently extracted, and subsequently treated with a UV-specific endonuclease to cleave all pyrimidine dimers. The sites of UV-specific nuclease scission within the centromere were determined by separating the DNA according to molecular weight, transferring the fragments to nitrocellulose, and hybridizing to a radiolabeled 624-bp fragment homologous to the centromere DNA from chromosome III. Several hotspots were identified in chromatin DNA from cells, as well as in irradiated deproteinized DNA. Double strand damage due to closely opposed pyrimidine dimers was also observed. At biological doses (35% survival) there are approximately 0.1 to 0.2 pyrimidine dimers per centromere. These dimers are efficiently repaired in the centromere and surrounding region.     

Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants

We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time. Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A+T-rich sequences in CDE II play important roles in mitosis and meiosis. Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation. In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation. Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently. The implications of these results for centromere function and chromosome structure are discussed.by M. Yanagida     

Role of conserved sequence elements in yeast centromere DNA.

Conserved sequence features in Saccharomyces cerevisiae CEN DNA are confined to a region of approximately 120 bp. The highly conserved 8 bp at the left (PuTCACPuTG) constitute the left boundary of a functional CEN DNA as shown by the analysis of a series of Bal31 deletions. The right boundary of a functional CEN DNA lies within the conserved 25 bp at the right (TGT-T-TG--TTCCGAA-----AAA) or a few base pairs further outside of the 120-bp region. One mutant which just lacks the left conserved DNA element PuTCACPuTG can still assemble into a partially functional mitotic centromere and it assembles into a well functioning meiotic centromere. The sequences between the two conserved terminal DNA elements can be increased in length (+50%) or in GC content (from 6% to 12%) without measurable changes in mitotic and meiotic segregations of plasmids carrying such CEN mutations. The naturally occurring length and GC content of this centromere DNA sequence element is, therefore, not essential for centromere function. We discuss the possibility that it partly acts as a hinge region between two domains. Finally, we tested integrations of CEN DNA into the genome and found a toleration of wild-type CEN6 DNA when present 3' of the LYS2 gene.     

Comparison of Hi-C results using in-solution versus in-nucleus ligation

BackgroundChromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation.ResultsWe show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries.ConclusionsWe conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0753-7) contains supplementary material, which is available to authorized users.     

Meiotic recombination within the centromere of a yeast chromosome

In order to examine the frequency of nonreciprocal recombination (gene conversion) within the centromere of the yeast chromosome, we constructed strains that contained heterozygous restriction sites in the conserved centromere sequences of chromosome III in addition to heterozygous markers flanking the centromere. One of these markers was the selectable URA3 gene, which was inserted less than one kb from the centromere. We found that meiotic conversion of the URA3 gene occurred at normal frequency (about 2% of unselected tetrads) and that more than one-third of these convertants coconverted the markers within the centromere. In addition, we observed tetrads in which conversion events extended through the centromere to include a marker on the opposite side from URA3. We conclude that meiotic conversion events occur within the centromere at rates similar to other genomic sequences.     

In vivo genomic footprint of a yeast centromere.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.     

PCR-based identification of wheat genomes

We describe a PCR system that distinguishes the A, B and D genomes in wheat DNA extracts. PCRs were directed at the ‘non-transcribed spacer’ regions of the rDNA loci. The spacers within the D genome locus have a 71-bp insertion that is absent from the corresponding A and B loci PCR product sizes therefore enable D- and D+ genomes to be distinguished. The A and B genomes can be differentiated by PCR with an internal primer which does not anneal to A genome sequences. This work is relevant to the ancient ecology of wheat, as it is often difficult to determine ploidy level from morphological examination of archaeobotanical remains.     

New method for yeast identification using Burrows-Wheeler transform

The explosive growth in biological data in recent years has led to the development of new methods to identify DNA sequences. Many algorithms have recently been developed that search DNA sequences looking for unique DNA sequences. This paper considers the application of the Burrows-Wheeler transform (BWT) to the problem of unique DNA sequence identification. The BWT transforms a block of data into a format that is extremely well suited for compression. This paper presents a time-efficient algorithm to search for unique DNA sequences in a set of genes. This algorithm is applicable to the identification of yeast species and other DNA sequence sets.     

Divergence in centromere structure distinguishes related genomes in Coix lacryma-jobi and its wild relative

Knowledge about the composition and structure of centromeres is critical for understanding how centromeres perform their functional roles. Here, we report the sequences of one centromere-associated bacterial artificial chromosome clone from a Coix lacryma-jobi library. Two Ty3/gypsy-class retrotransposons, centromeric retrotransposon of C. lacryma-jobi (CRC) and peri-centromeric retrotransposon of C. lacryma-jobi, and a (peri)centromere-specific tandem repeat with a unit length of 153 bp were identified. The CRC is highly homologous to centromere-specific retrotransposons reported in grass species. An 80-bp DNA region in the 153-bp satellite repeat was found to be conserved to centromeric satellite repeats from maize, rice, and pearl millet. Fluorescence in situ hybridization showed that the three repetitive sequences were located in (peri-)centromeric regions of both C. lacryma-jobi and Coix aquatica. However, the 153-bp satellite repeat was only detected on 20 out of the 30 chromosomes in C. aquatica. Immunostaining with an antibody against rice CENH3 indicates that the 153-bp satellite repeat and CRC might be both the major components for functional centromeres, but not all the 153-bp satellite repeats or CRC sequences are associated with CENH3. The evolution of centromeric repeats of C. lacryma-jobi during the polyploidization was discussed.