Mechanisms for histone segregation at DNA replication forks and implications for epigenetic inheritance.

Epigenetic information encoded in chromatin is responsible for sustaining transcriptional programs that determine cell identity and regulate differentiation. In every mitotic cell division, this epigenetic information must be faithfully transmitted to daughter cells to preserve specific transcriptional landscapes. Failure in this process compromises the maintenance of cell identity and can lead to tumorigenesis. Histone posttranslational modifications control the interactions between DNA and other proteins, like transcription factors, and are therefore considered epigenetic marks. However, how these histone modifications, present at parental nucleosomes, are transferred to both leading and lagging strands during DNA replication remains largely unknown. Using the novel method eSPAN to determine strand-specific protein enrichment at replication forks, we have recently shown how two non-essential subunits of leading strand DNA polymerase ? and the Mcm2-Ctf4-Pol ? axis mediate parental histone transfer to leading and lagging strands, respectively. These important findings indicate that a symmetric inheritance of epigenetic information between daughter cells requires the action of several different factors. However, mutations at these known factors impacting parental histone transfer have a minor effect on cell viability. Therefore, I hypothesize that a complex network of proteins yet to be discovered regulates this process. This project will focus on the identification and characterization of all those factors involved in the segregation of parental histones to nascent DNA strands using a combination of genetic, biochemical and genome approaches. Our first aim will be to determine how the conserved master regulator of replication PCNA functions in deposition of parental nucleosomes to lagging strands (Aim 1). Our second aim is to identify and characterize additional factors playing a role in the transmission of epigenetic information to daughter DNA strands using a combination of candidate and genome wide screen approaches (Aim 2). Moreover, we will adapt the eSPAN method to investigate the re- establishment of heterochromatin on newly synthesized DNA strands to delve further into the relationship between asymmetric parental nucleosome segregation and loss of transcriptional silencing. Taken together, this project will shed light into the underlying mechanisms of epigenetic inheritance, a critical but poorly understood process involved in several diseases like cancer. Aim 1 will be started and completed during the K99 phase under the mentorship of Dr. Zhiguo Zhang, a leader in the field of histone epigenetics, at Columbia University, an outstanding research institution. Aim 2 will start at the end of K99 phase but will continue during the R00 independent phase. The funds and support from this award will allow me to focus on my research while giving me the opportunity to learn about laboratory management, technology development, and data analysis, critical skills to become a successful independent researcher.