1. Lindsey M. Maurice-Walker
June 2, 2014
Role Determination of the Chromatin Assembly Factor-1 (CAF1) in Epigenetic Processes
Chromatin assembly and disassembly during DNA replication requires several
interactions between replication proteins, chromatin assembly factors, and histone
modification proteins. The coordination of these proteins is important for reformation of
preexisting epigenetic states and propagation of chromatin modifications (Jacobi, 2006).
Also, studies have observed a direct link between phosphorylation of some of these
proteins and mechanisms associated with DNA repair (Moggs, 1999). In the lab, we use
Saccharomyces cerevisiae, budding yeast, as a model to understand how genes are
expressed and regulated in the cell. Yeast is not only a viable organism to study because
of its rapid growth and versatile transformation system, but the proteins involved in
replication and altering chromatin structure in yeast (i.e. Asf1p, Cdc7-90, and CAF-1) are
highly conserved in eukaryotes. One of the proteins involved in replication, gene
regulation, and DNA repair is Chromatin Assembly Factor 1 (CAF-1), a histone
chaperone. During replication, CAF-1 transports newly synthesized histone H3/H4
tetramers to the replication fork. Chromatin Assembly Complex-1 (Cac1), a subunit of
CAF-1 in yeast, binds to newly synthesized histone dimers, and interacts directly with the
Proliferating Cell Nuclear Antigen (PCNA), a DNA polymerase processivity factor. A
previous study has shown that the interaction between PCNA and the p150 subunit
(human ortholog of Cac1) of CAF-1 is regulated by the phosphorylation of p150 by the
replication kinase Cdc7-Dbf4 (Gerard et al., 2006). This mechanism is essential for
histone deposition during DNA replication, and serves as a control point that can regulate
other DNA repair pathways. p150 exhibits similar functions in mammalian cells as Cac1
does in yeast cells. Therefore, understanding how CAF-1 is regulated and how its
regulation influences chromatin structure, gene expression, and DNA repair processes
will provide insight into how these mechanisms operate in mammalian cells, which could
potentially develop into effective, new drug-targeting therapies.
In addition to regulating CAF-1’s interaction with PCNA, we hypothesize that the
phosphorylation of CAF-1 could also regulate its role in gene silencing and response to
DNA damage as well. To test this, we grow strains of Saccharomyces cerevisiae in the lab
with deleted CAC1 and/or mutated CDC7 genes (cac1Δ and cdc7-90). Once the cells are
grown, we transform them with a plasmid that contains point mutations of the CAC1
gene. The plasmids we use during transformation also contain the URA3 gene that allows
the cells to grow in the absence of uracil and is used as a marker to select by growth for
transformed cells.