However, in eukaryotes, genome-wide nucleosome positioning

does not appear to be dictated solely by DNA sequence, as the addition of ATP to chromatin incubated in whole cell extracts is necessary to recapitulate nucleosome phasing in vitro, indicating that ATP-dependent chromatin remodelers play an Apoptosis Compound Library concentration important role in defining nucleosome positions within the cell [ 29]. Yet, other studies have highlighted the importance of AT-rich DNA sequences in maintaining NDRs in vivo [ 30 and 31]. Thus, while the primary sequence of DNA does position nucleosomes in select locations in the genome, trans-acting factors play an equally significant role in over-ruling intrinsic DNA-sequence based nucleosome positioning. Together, evolutionary conserved nucleosome positioning coupled to ATP-driven chromatin remodelers provide a powerful one-two punch, permitting chromatin structure to be flexible and responsive to changing environmental cues from the cell. Despite decades of nucleosome positioning research, surprisingly little information is available on the interplay between key histone variants and nucleosome positioning. Using a 208 bp fragment of DNA, it is apparent simply from monitoring the ABT 737 migration of the nucleosomes through a native gel that the histone variants H3.3 and H2A.Z both modify the position of the nucleosome upon the DNA in vitro

[ 20]. However, no extant study has yet undertaken the difficult yet exciting task of investigating whether individual histone variants, which are all at subsaturating levels in vivo, manipulate structural motifs within DNA sequences to potentially out-compete other histone variants for certain positions in the genome, or to create specialized chromatin many structures that are co-dependent on the presence of the histone variant and the sequence of the underlying DNA. While histone

variants play an important role in regulating gene expression, they may also participate in their own epigenetic inheritance, maintaining correct localization on the newly synthesized daughter strands following DNA replication. Using a SILAC-based (stable isotope labeling by amino acids in cell culture) approach, it was recently determined that after two cell cycles, ∼20% of the core (H3.3/H4)2 tetramer within nucleosomes were split into H3.3/H4 dimers, assembled with newly synthesized H3.3/H4 [32]. These data support a model in which segregated deposition of parental H3.3/H4 after DNA synthesis is responsible for maintaining the local epigenetic state (Figure 2a) [33]. The splitting process appears to be primarily replication-dependent, as treatment with hydroxyurea or aphidicolin significantly reduced splitting events. In contrast, the remaining (H3.3/H4)2 tetramers, along with the canonical (H3.