The assembly of chromatin is a necessary process that must occur following DNA-utilizing processes, such as replication, repair, and transcription. Chromatin is a complex of DNA and proteins that is responsible for packaging and organizing DNA into the eukaryotic nucleus and also serves as a regulator of all DNA-utilizing processes. The basic repeating unit of chromatin is the nucleosome, which consists of approximately 147 base pairs of DNA wrapped around a histone octamer core. Chromatin assembly is facilitated by core histone chaperones and ATP -dependent motor proteins. For this dissertation, I have investigated the mechanism of chromatin assembly using a purified, defined in vitro chromatin assembly system, consisting of relaxed DNA, ATP, core histones, the histone chaperone NAP1, and the ATP-driven motor proteins ACF or CHD1. A novel nonnucleosomal histone-DNA complex, termed the prenucleosome, was identified as an intermediate of chromatin assembly. Whereas this intermediate of chromatin assembly is biochemically distinct from canonical nucleosomes, the prenucleosome is indistinguishable from a nucleosome by atomic force microscopy. Analysis of chromatin remodeling-defective mutants of CHD1 demonstrated that ATP-dependent chromatin assembly is a distinct activity from chromatin remodeling. These findings indicate a multi-step mechanism for chromatin assembly. Histone chaperones first deposit histones onto DNA to generate prenucleosomes. These prenucleosomes are subsequently converted into randomly-positioned canonical nucleosomes by ATP-dependent motor proteins. Lastly, ATP- driven motor proteins reposition these nucleosomes into periodic arrays. Altogether, these studies provide new insight into the mechanism of ATP-dependent chromatin assembly, highlighting the necessity of ATP-driven motor proteins in this process

Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly. DOI:http://dx.doi.org/10.7554/eLife.00863.001.

Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼ 80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone-DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone-DNA species in the cell.

Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Krüppel-like factor 4 (Klf4) expression in naïve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two naïve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying naïve pluripotency.