Browsing by Subject "Replication timing"
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Item Open Access DNA Replication of the Male X Chromosome Is Influenced by the Dosage Compensation Complex in Drosophila melanogaster(2013) DeNapoli, LeynaAbstract
DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.
DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.
In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.
The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.
We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).
Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome.
We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.
In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.
In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program.
Item Open Access Re-replication in the Absence of Replication Licensing Mechanisms in Drosophila Melanogaster(2011) Ding, QueyingTo ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability.
DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in Drosophila Menalogaster , is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4Ddb1 mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase.
In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.
In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.