The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes

Abstract

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. We show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.Chromatin-remodeling motors play essential roles in organizing the chromatin state for regulating eukaryotic genomes, yet how they carry out their myriad activities is poorly understood. Their substrate, the nucleosome, contains 147 bp of DNA wrapped in ~1.5 turns Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms around an octamer of histone proteins. Even the smallest movement of the histone octamer relative to the DNA presumably requires a coordinated process of breaking and reforming the many histone-DNA contacts. The ACF chromatin-remodeling complex exemplifies the task, as it is able to move nucleosomes to create evenly spaced nucleosomal arrays that contain equal DNA on either side of each nucleosome1-10. These evenly spaced arrays are important for packaging the underlying DNA into silent chromatin structures in vivo1-10. HHS Public AccessACF is part of the ISWI family of remodeling complexes. The ATPase subunits of ISWI complexes can move nucleosomes by themselves while the accessory subunits modulate this basic activity11-15. The human ACF complex consists of one ATPase subunit, SNF2h and one accessory subunit, Acf16,7. SNF2h is part of the SF2 family of DExx box proteins that includes helicases and nucleic acid translocases16. The ATPase domain of SNF2h has two RecA-like domains, which are thought to form a cleft within which ATP binds. SNF2h also has an alpha-helical extension comprised of three additional domains, HAND, SANT and SLIDE which are thought to play a role in bi...

Sequence-based prediction of single nucleosome positioning and genome-wide nucleosome occupancy

“…In order to maintain a controlled distribution of distances, nucleosomes need to be repositioned, most likely by ATP-dependent chromatin-remodeling enzymes such as ACF and CHD1 (65)(66)(67)(68)(69). Catalyzed nucleosome repositioning has been the subject of numerous experimental and theoretical studies (5,(70)(71)(72)(73)(74) but the exact mechanism by which these proteins move the histones along the DNA sequence remains unclear, partially because the mechanisms of remodelers can be heavily convoluted with nucleosome positioning effects that are encoded in the DNA.…”

Nucleosome positioning in a model of active chromatin remodeling enzymes

“…Nucleosome adsorption is known to occur efficiently in vivo by ATP-independent histone chaperone-mediated pathways; here we consider only ATP-independent nucleosome adsorption. On the other hand, thermally excited desorption and sliding are very slow, and ATP-dependent enzymes are known to assist nucleosomes in these processes (11)(12)(13)(14)(15)(16)(17). We consider both ATP-dependent and ATP-independent desorption and sliding processes.…”

“…However, disrupting nucleosomes requires passage of energy barriers of tens of k B T per nucleosome (7,8). In vivo this is facilitated by ATP-consuming "chromatin remodeling complexes" (9,10), which catalyze nucleosome repositioning and disassembly (11)(12)(13)(14)(15)(16)(17).…”

“…In addition to thermal fluctuation dynamics (always present as a "background" to any active remodeling machinery) we consider two general types of remodeling machines that: (i) slide nucleosomes along the DNA to cause repositioning, and (ii) remove (eject) histone octamers from DNA. These two processes have been implicated to be catalyzed by remodelers in the ISWI [e.g., yeast ISWI (14), ACF (17)] and SNF2 [e.g., RSC (11), yeast SWI/SNF (12,15)] subfamilies, respectively. Using experimentally determined thermal and ATP-dependent enzyme rates we show that active sliding is necessary to achieve biological densities of nucleosomes within biologically relevant time scales, but that active nucleosome removal is also needed, to obtain nucleosome positioning at biological densities.…”

Principles and practice of nucleosome positioningin vitro

“…This FRET response is highly sensitive over a window around the characteristic Förster radius of the dye pair, which is typically in the range 10-100 Å. spFRET can be performed in solution (Tóth et al 2001), or particles can be spread on an imaging surface and the relative signal of the FRET dyes observed for each single molecule spot simultaneously for thousands of spots with high time resolution (Koopmans et al 2007). spFRET revealed rates of site exposure of DNA from the edge of the nucleosome (Li et al 2005) and quantitation of nucleosome sliding (Racki et al 2009). …”