The chromatin environment shapes DNA replication origin organization and defines origin classes
How do chromosomes, DNA and genes all fit together? The chromatin further loops and coils to form the tightly condensed chromosome We can use the analogy of a city to better understand the relationship between DNA molecules. The kinetochore is essential for high-fidelity chromosome segregation in every The features of a kinetochore include centromeric (CEN) DNA, specialized protein .. the motif-based sequence analysis software MEME (fim-mdu.info net). .. The kinetochore and the centromere: a working long distance relationship. Programmed DNA double-strand breaks (DSBs) are required for meiotic Within the elongated nucleus, chromosomes are arranged in a a conserved motif search in Pars11 using MEME (66) did not reveal the .. Structure of the germline genome of Tetrahymena thermophila and relationship to the.
Yet, neo-X genes are transcriptionally more active in males, relative to females, suggesting the evolution of incipient dosage compensation on the neo-X. Our data show that Y degeneration proceeds quickly after sex chromosomes become established through genomic and epigenetic changes, and are consistent with the idea that the evolution of sex-linked chromatin is influenced by its ancestral configuration.
Author Summary DNA is packaged with proteins into two general types of chromatin: Sex chromosomes typically evolve from a pair of euchromatic autosomes.
The Y chromosome of Drosophila is gene poor and almost entirely heterochromatic; the X chromosome, in contrast, has evolved a hyperactive euchromatin structure and globally up-regulates its gene expression, to compensate for loss of activity from the homologous genes on the Y chromosome.What is a Chromosome?
The evolutionary trajectory along which sex chromosomes evolve such opposite types of chromatin configurations remains unclear, as most sex chromosomes are ancient and no longer contain signatures of their transitions. Gene expression is lower on the neo-Y than on the neo-X, which is associated with a higher level of binding of a silencing heterochromatin mark.
The neo-X, on the other hand, shows no evidence of evolving hyperactive chromatin for dosage compensation. Our results show that the Y chromosome can degenerate quickly, but the tempo and mode of chromatin evolution on the sex chromosomes may be constrained by the ancestral chromatin configuration. Introduction Sex chromosomes have originated independently many times from ordinary autosomes in both plants and animals [ 1 ].
A common feature of heteromorphic sex chromosomes is that while X chromosomes maintain most of their ancestral genes, Y chromosomes often degenerate due to their lack of recombination, with only few functional genes remaining for a recent review see [ 2 ].
The loss of gene function is often accompanied by an accumulation of repetitive DNA on ancient Y chromosomes, and a switch of chromatin structure from euchromatin to genetically inert heterochromatin [ 23 ]. Loss and silencing of Y-linked genes drives the evolution of dosage compensation on the X chromosome, which is often mediated by chromosome-wide epigenetic modifications.
Studies of young sex chromosomes have improved our understanding of the genomic and epigenomic mechanisms driving the divergence between X and Y [ 6 — 9 ]. Neo-sex chromosomes of Drosophila are formed by chromosomal fusions between the ancestral sex chromosomes and ordinary autosomes. The neo-Y, which is the autosome that became linked to the Y, entirely lacks recombination since it is transmitted through males only, which in Drosophila do not undergo meiotic recombination.
Consistent with theoretical predictions that selection is ineffective on non-recombining chromosomes [ 10 ], neo-Y chromosomes in several Drosophila taxa have undergone chromosome-wide degeneration, and the extent of gene loss roughly corresponds to the age of the neo-Y.
This is in agreement with the hypothesis that this G-rich nucleosome-free region may be the pre-RC assembly site. Interestingly, Saccharomyces japonicus are characterized by high GC content, while AT content is a negative predictor of origin function Xu et al.
As G4 can inhibit replication fork progression Sarkies et al.
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DNA replication initiation occurs in two temporal steps. In their folded form, G4 could negatively regulate initiation of DNA replication at pre-RCs; however, when unfolded, they could trigger DNA replication at the subpopulation of pre-RCs that are activated during S phase.
G4 formation may be favored by formation of an R-loop on the C strand Fragkos et al. Alternatively, replication origins could be activated at pre-RCs and then transiently stall at the proximal G4 on the leading strand, a phenomenon similar to transcriptional pausing Liu et al. This mechanism could be used to start initiation on the opposite strand, similarly to the model proposed for the DBF4 replication origin Romero and Lee Alternatively, it might control the timing of origin activation, as Rif1 has recently been found to bind G4 Kanoh et al.
This is reminiscent of the nucleosome positioned downstream from the ORC binding site described in budding yeast origins Eaton et al. In agreement, we found a strong enrichment of H3K64ac strictly centered on the nucleosome at the IS, a new chromatin mark associated with labile nucleosomes in mESCs Di Cerbo et al.
The chromatin environment shapes DNA replication origin organization and defines origin classes
These results suggest a new role for this mark in positioning a labile nucleosome that prevents the accessibility to the IS outside the required time and that can be remodeled when the pre-RC is activated. Each class is characterized by a specific origin structure and relationship with the surrounding chromatin.
Class 2 origins correspond to large initiation zones with many inefficient initiation sites and are overrepresented in enhancer regions identified by H3K4me1possibly offering more flexibility to initiate DNA replication. Conversely, strong SUZ12 enrichment was found in the highly efficient Class 3a origins that replicate early in S phase.
Moreover, loss of SUZ12 Pasini et al. The explanation given was that DNA replication fork stalling promotes the activation of dormant origins. The partial effect on inter—origin distances observed by Piunti et al.
These observations suggest that PcG proteins structurally restrict the position of origin activation inside Polycomb domains. The high efficiency of Class 3a origins and the absence of any other strong origin in the vicinity imply a constraint to initiate replication at specific locations in these regulatory regions.
Genetic and epigenetic guidance of replication initiation sites Our analysis shows that combinations of specific genomic signatures regulate the localization of IS in complex eukaryotes.
Whats the difference between Chromatin and a Nucleosome? : biology
Importantly, specificity appears to be achieved by the synergic action of sequence motifs and chromatin modifications to allow the flexible selection of replication origins in different genomic contexts. In gene-poor regions that are replicated late in the cell cycle, origin selection seems to be more dictated by sequence-specific features. Conversely, epigenetic signatures appear more involved in origin selection in early replicating gene-rich regions, in agreement with their open chromatin configuration.
Our study also shows that IS are characterized by specific signatures, such as a well-positioned nucleosome with specific sequences and epigenetic marks. In contrast, the upstream sequence contains a G-rich element inside a nucleosome-depleted region, which is likely to be the pre-RC site.
The existence of several classes of origins, with distinct sets of features to control origin positioning, appears to be an important mechanism to regulate origin usage. This flexibility in elements that can drive origin positioning could be used by eukaryotic cells to adapt to the environment, to the constraints linked to the complex structure and variety of conformations of chromatin and chromosomes, and finally to different tissue-specific transcription programs.
Nascent strand isolation Nascent strands NS were purified as previously described in detail Cayrou et al. The resulting DNA was precipitated with ethanol, phosphorylated again, and digested again in the same conditions.
What is the relationship of DNA, a chromosome, and chromatin?
Three samples were purified from three independent cell cultures. This protocol differs in several ways from a recently described method Foulk et al. Instead, a large amount of whole replicating DNA was used. This is to fold less than in our previous works Supplemental Fig. S2; Cayrou et al. For each sample, sequenced reads were mapped as in Supplemental Material.
Peak calling After a thorough evaluation Supplemental Materialwe combined two peak calling programs with complementary properties Fig. Random peak selection As a negative control for the subsequent analyses peak clustering, coverage of chromatin marks, motif discoverythe RSAT random-genome-fragments program was used to select random genomic regions of the same number and sizes as the origin peaks. Read density maps Read occurrence profiles were obtained by extracting 7 kb on each side of the summit of each peak kb data provided in Supplemental Material and counting the reads per bp bins.
Data processing was done in R R Core Team by applying k-means clustering on the read occurrence profiles with various numbers of clusters. For visual purposes, the resulting clusters were sorted according to their average density profile to highlight the relationships between the central peak and its first closest neighbor. On the density map, pairs of neighbor peaks appear twice: Consistently, our clustering procedure revealed symmetrical clusters.
For further analysis, we regrouped the symmetrical pairs of clusters e. We developed a custom R library ocpR; available in Supplemental Materials and http: Clustering results with other k values are in Supplemental Material.
Overlapping of origins with the genomic annotations was done as in the Supplemental Material. The accession numbers are detailed in Supplemental Table S1.