DNA in eukaryotic cells is organized into chromatin. Far from being an inert packing material, chromatin is a rich source of information for gene regulation. Some of this information, including post-translational modification of histones, is believed to be propagated through cell division to mediate epigenetic memory of transcriptional states. Each time cells pass through S-phase, every nucleosome in the genome is disrupted. Furthermore, half of the histones deposited on the duplicated genome are newly synthesized, and lack histone modifications linked to gene regulatory events. Thus, whether and how histones can carry epigenetic information about gene expression patterns through DNA replication remains incompletely resolved After DNA replication, histone modifications are diluted two-fold, which could result in loss of epigenetic information. After DNA replication, the two sister chromatids are held together through the action of the cohesin complex. One way to buffer the effect of diluting the histones is if histone-based information can be “shared” through the pairing of sister chromatids. To test this idea, we will analyze the rate and extent of restoration of histone modifications in cells with disrupted sister pairing. The recognition of histone modifications by the enzymes that create them is believed to be important for re-establishing histone modifications after DNA replication (sometimes referred to as a read-write mechanism). In principle, this mechanism could function both in cis and in trans (i.e. between sisters). We will use in vitro experiments ranging from simply tethering modified and unmodified templates to in vitro replication in the presence or absence of sister cohesion to test this model. Together, the in vitro and in vivo approaches will test the idea that sister chromatids exchange epigenetic information to maintain chromatin states.
This project involves genome engineering, and analysis of chromatin in vivo (e.g. ChIP) in cultured cells. It also depends heavily on in vitro reconstitution of biochemical reactions, including histone modification and DNA replication. This aspect of the project will require protein purification, chromatin assembly, and extract preparation (from Xenopus eggs).
Background in molecular biology and biochemistry or biophysics is highly recommended, but the most important characteristics are motivation, ability to work independently, and willingness to learn. We would also welcome students with more theoretical or computational backgrounds.
Madamba, E.V., Berthet, E.B., and Francis, N.J. (2017) Inheritance of histones H3 and H4 during DNA replication in vitro. Cell Reports 5: 1361-1374 PMID 29091772.