Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS.

Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level (∼100-1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.

A cell-free CENP-A assembly system defines the chromatin requirements for centromere maintenance.

Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes.

CENP-A confers a reduction in height on octameric nucleosomes.

Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.

A cell-free system for functional centromere and kinetochore assembly.

This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d.

CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly.

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

In vitro centromere and kinetochore assembly on defined chromatin templates.

During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.

Local geometry and elasticity in compact chromatin structure.

The hierarchical packaging of DNA into chromatin within a eukaryotic nucleus plays a pivotal role in both the accessibility of genomic information and the dynamics of replication. Our work addresses the role of nanoscale physical and geometric properties in determining the structure of chromatin at the mesoscale level. We study the packaging of DNA in chromatin fibers by optimization of regular helical morphologies, considering the elasticity of the linker DNA as well as steric packing of the nucleosomes and linkers. Our model predicts a broad range of preferred helix structures for a fixed linker length of DNA; changing the linker length alters the predicted ensemble. Specifically, we find that the twist registry of the nucleosomes, as set by the internucleosome repeat length, determines the preferred angle between the nucleosomes and the fiber axis. For moderate to long linker lengths, we find a number of energetically comparable configurations with different nucleosome-nucleosome interaction patterns, indicating a potential role for kinetic trapping in chromatin fiber formation. Our results highlight the key role played by DNA elasticity and local geometry in regulating the hierarchical packaging of the genome.