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Deciphering the roles of chromatin modifiers in germline aging

Periodic Reporting for period 1 - Egg-Juvenate (Deciphering the roles of chromatin modifiers in germline aging)

Reporting period: 2015-05-01 to 2017-04-30

Faithful transfer of genetic material is critical for the success of all cell divisions. Yet as the organism ages this process becomes more error-prone, leading to aneuploidic daughter cells due to mis-segregation of chromosomes. This can lead to cancer in mitotic cells and in the female germline it leads to reduced fertility and an exponential increase in miscarriages and birth defects already at the third decade of life. In humans, all the oocytes begin their development in-utero but arrest for decades in the middle of this process, leading to the rapid increase in meiotic divisions failure. The oocytes arrest while undergoing dramatic structural changes, termed chromosome remodeling. Very little is known about what drives this critical phase in germline development, yet it is assumed to involve chromatin modifiers and DNA binding complexes. We suggested to identify chromatin-modifying complexes that control oogenesis progression and find their mechanism of operation. The research was aimed to find the major players in this processes and their mode of operation. Given that maternal age is rising in the western world, translational implementation of this research would have a dramatic impact on our scientific understanding of fertility.
The conclusions of the work provided insights on the major part of the MAPK biochemical pathway plays in the chromosomal separation which are at the core of oogenesis, and novel master regulators that work through this pathway.
According to our research plan we identified a gene called ogr-2 that we suspected may have roles in oocyte formation. Specifically our preliminary work showed that depleting the expression of this gene prevents correct chromosomal structure in the mature oocyte. We engineered a worm strain without this gene by deleting its sequence from the genome using the CRISPR/Cas9 technology. Our unique approach verified a null allele, by executing a directed DNA double strand breaks at targeted sites before and after the gene. To ensure this engineering was not accompanied by any off target effect we outcrossed our strain 5 times. In this strain we found problems in the oogenesis progression and the chromosomes in mature eggs had aberrant formation. We found that the timing in which germ cells are moving from proliferating into division stage is delayed in the mutant. Moreover we found that the structure of the chromosomes in the mature oocytes is abnormal, and shows multiple chiasma, indications that the crossover interference was relieved. We found a statistically significant increase in the number of programmed cell death among meiocytes in the mutant. Surprisingly this death was not due to the canonical checkpoints, synapsis and double strand breaks repair, but instead was due to activation of MAPK, a major biochemical pathway, at the wrong developmental stages. This pathway is normally activate at two time points: mid-pachytene and late diakinesis. In the mutant is was activated in the proliferative region and diplotene. This can explain the changes listed above.

We presented these results in several meeting and we are currently writing a manuscript to be submitted to peer reviewed journal.
Beyond the state of the art, our work identified a novel major master regulator of oogenesis. This highlights a new aspect by which the entire oogonial processes is controlled and timed. Moreover, our work focused the efforts into the specific biochemical pathway that control the timing of oogenesis timing and aging. We can now revisit long standing questions from this new perspective, which bypass the old concepts of canonical checkpoints.

This work opens new aspects of our understanding of how oocytes are formed, and we believe it will help in fertility research.