A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.

Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4.

A dynamic interplay of enhancer elements regulates expression in naïve pluripotency.

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 -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 () expression in naïve pluripotency.

Nucleosomes impede Cas9 access to DNA in vivo and in vitro.

The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9).

The prenucleosome, a stable conformational isomer of the nucleosome.

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.

ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling.

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.

Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF.

Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF.

A class II histone deacetylase acts on newly synthesized histones in Tetrahymena.

Newly synthesized histones are acetylated prior to their deposition into nucleosomes. Following nucleosome formation and positioning, they are rapidly deacetylated, an event that coincides with further maturation of the chromatin fiber. The histone deacetylases (HDACs) used for histone deposition and de novo chromatin formation are poorly understood.