Science Spotlight

Fire away: chromatin context influences timing and efficiency of origin firing

From the Tsukiyama Laboratory (Basic Sciences Division)

The genetic information in our cells is stored in an elegant double-helical molecule of DNA that is wrapped around protein complexes called nucleosomes to form chromosomes.  Life depends upon the successful replication of DNA as well as the maintenance of the nucleosomes that the double helix wraps around.    DNA replication is initiated at distinct locations in chromosomes called origins of replication. Biologists say that an origin "fires" when DNA replication begins there.  In each human cell, there are thousands of origins of replication and the timing and efficiency of the firing of each origin varies.  While the exact sequence and position of replication origins in human cells is not very well understood, the origins of the single-celled model organism Saccharomyces cerevisiae (AKA budding yeast) have a short, characteristic DNA sequence.  It has been postulated that the presence of many origins enables the rapid replication of DNA but it has been found that not all of them actually fire or fire at the same time in a given cell.  Even in budding yeast where origins are sequence-defined, not all origins actually fire in a given round of replication.  The mystery of what determines whether an origin will fire is ongoing.  In their recent publication in Genome Research, scientists in the Tsukiyama Laboratory (Basic Sciences Division) found that the timing and efficiency of origin firing in budding yeast was strongly correlated with the nucleosome occupancy in the surrounding DNA.  

Diagram illustrating the concept of nucleosome occupancy. Nucleosome occupancy is a measure of how likely it is to find a nucleosome at a given position in the genome. Graphic courtesy of Toshi Tsukiyama

Origins of replication are found in regions of DNA with fewer nucleosomes, called nucleosome-depleted regions (NDRs), and the nucleosomes surrounding the origins have been found to sit at well-defined positions.  However, the positions of nucleosomes in the DNA varies from cell to cell and the overall likelihood of finding nucleosomes surrounding a given origin of replication has not been systematically analyzed and may define the ability of a given origin to fire.  This frequency of the presence of a nucleosome at a given position in the DNA, called nucleosome occupancy, can be measured by determining the strengths of nucleosome signals in the genome of a population of cells. Scientists in the Tsukiyama Lab measured nucleosome occupancy by performing chromatin immunoprecipitation of the nucleosome protein histone H3 following sonication of the DNA input.  By sonicating the input DNA rather than the traditional method of digestion using micrococcal nuclease (MNase), the occupancy of the nucleosomes surrounding NDRs can be more accurately measured because nucleosomes surrounding NDRs are hyper-sensitive to MNase.  By measuring nucleosome occupancy genome-wide in a population of budding yeast, the scientists were able to sort the 798 predicted origins into classes of low, medium, and high nucleosome occupancy.  

The order of firing of the origins in the yeast genome has been measured by treating the cells with low doses of a drug, hydroxyurea, which depletes free nucleotides needed for replication.  Only early-firing origins fire under this condition.  Origins also vary in their efficiency, which reflects the probability by which any given origin fires in any given cell-cycle.  The scientists found a statistically significant correlation between low nucleosome occupancy and early, more efficient origin firing. 

The scientists wanted to determine whether nucleosome occupancy surrounding origins is different before versus after cells have committed to replicate, in other words whether it is cell cycle regulated. To do this they measured nucleosome occupancy in cells in G1, which is the state preceding DNA replication, and in G2/M phase, which immediately follows replication.  Interestingly, they found that origins bound with pre-replication components (the ORC complex) had much higher nucleosome occupancy in G2/M versus G1.  In fact, the level of nucleosome occupancy surrounding these origins is about 2-fold lower in G1 compared to G2/M.  Furthermore, they found that origins with lower nucleosome occupancy in G1 had relatively greater loss of nucleosomes between G2/M to G1 and vice versa.  These results suggest that nucleosome occupancy is established between G1 and G2/M, during DNA replication in S phase.

Overall, their results suggest that the occupancy of nucleosomes surrounding origins is regulated across the cell cycle and by pre-replication components.  The relative nucleosome occupancy of a given origin affects its ability to fire and support the replication process.  Therefore, the regulation of nucleosome incorporation and positioning is an essential process to maintain cell viability. 

Rodriguez J, Lee L, Lynch B, Tsukiyama T. 2016.  "Nucleosome occupancy as a novel parameter for replication origin functions."  Genome Research.  Epub ahead of print.  

This research was funded by the National Institutes of Health and a Cellular and Molecular Biology Training Grant.  


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