Epigenetic mechanisms specify cell fate decisions by altering chromatin structure and gene expression patterns while preserving primary DNA sequences. Epigenetic mechanisms play a key role in specifying and maintaining stem cell identity throughout multiple cell divisions. Many types of stem cells have the ability to asymmetrically divide to give rise to one daughter cell capable of self-renewal and another daughter capable of differentiating. Previously, we discovered that the preexisting H3 is segregated to the male germline stem cell (GSC) whereas newly synthesized H3 is enriched toward the differentiating daughter cell in Drosophila melanogaster. Since post-translational histone modifications are a key component of the epigenome, our studies provide the first direct evidence suggesting that stem cells may selectively retain preexisting histones that define their stem cell identity. We have demonstrated that this asymmetric pattern is specific to the canonical H3, but not for histone variant H3.3. Because H3 is incorporated during DNA replication whereas H3.3 is incorporated in a replication-independent manner, our findings suggest a potential role for DNA replication in establishing epigenetic information.
Using super-resolution microscopy, I have been able to study the localization patterns of preexisting vs. newly synthesized histones for histones H3 and H3.3. Whereas histone H3.3 shows no separation between preexisting and newly synthesized histones during DNA replication, histone H3 shows regions of separation approximately 200nm in size. Furthermore, using a technique known as a proximity ligation assay (PLA), I have been able to test whether different histone populations show a strand preference (leading vs. lagging) during their incorporation onto nascent chromatin. By testing the proximity of preexisting and newly synthesized histones to proteins known to be enriched upon the lagging strand, I have generated preliminary data suggesting that newly synthesized histones are preferentially incorporated onto the lagging strand. In addition, I have been able to nucleotide analogues (EdU, BrdU) to observe regions of DNA replication in the Drosophila germline. By comparing the replication patterns observed in asymmetrically dividing GSCs to the replication patterns observed in symmetrically dividing progenitor germ cells, I have been able to generate preliminary data suggesting that GSCs coordinate DNA replication in a manner distinct from symmetrically dividing progenitor cells. Based on these data, we propose that GSCs may regulate DNA replication in such a manner that sister chromatids may be built primarily via leading-strand synthesis or lagging-strand synthesis, thereby coordinating the differential deposition of preexisting vs. newly synthesized histones onto distinct sister chromatids.