Cairns Lab - Chromatin Remodeling Mechanisms

To understand the mechanism that remodelers use to move or eject nucleosomes—we purify remodeler complexes and examine their action on purified nucleosomes in vitro, and collaborate to reveal their high-resolution structures, and dysregulation in cancer.

Mobilizing Nucleosomes—The Basic Unit of Chromatin

Chromatin remodelers are fascinating machines that slide or eject nucleosomes, which is the fundamental packaging unit of chromosomes. Nucleosomes can block the binding of transcription factors, and therefore their repositioning or removal can enable factors to bind to the exposed DNA, to enable gene expression or DNA repair. The primary remodeler that we have studied is called RSC (remodels the structure of chromatin), a SWI/SNF-subfamily remodeler (Kasten et al., Cell 2011). It consists of 15 proteins in one large complex that uses the energy of ATP hydrolysis to slide and eject nucleosomes to help transcription factors bind DNA. We have also made major mechanistic contributions to an alternative type of remodeler, termed ISWI, which achieves an opposite outcome—ISWI helps organize and properly space nucleosomes to block transcription factor binding (Clapier et al., Nature 2012).

A main discovery of our lab is the mechanism for how ATP hydrolysis moves and ejects nucleosomes. We discovered that both remodelers contain an "engine/motor" that burns ATP as a fuel to conduct DNA translocation (Saha et al., Genes and Development 2002) This DNA translocase motor binds within the nucleosome, and pumps DNA around the surface of the nucleosome in the form of DNA waves, resulting in the movement of the histone octamer relative to the DNA (Saha et al., Nature SMB 2005). The key to understanding how regulated DNA translocation leads to gene regulation is described below. We have also collaborated with Carlos Bustamante (HHMI, UC Berkeley) and Yongli Zhang (Yale University) to determine important biophysical parameters of DNA translocation, such as the speed and force of DNA translocation on individual RSC-nucleosome complexes.

Structures of Chromatin Remodelers with the Nucleosome

To provide structural models for chromatin/nucleosome remodeling, we initially collaborated with Eva Nogales (HHMI, University of California, Berkeley) and Andres Leschziner (UC San Diego) to determine the EM structure of the entire RSC complex at low-moderate resolution, which revealed a large flexible protein machine that contains a large pocket of nucleosome dimensions. More recently, we have collaborated with Zhucheng Chen at Bejing University to solve the high-resolution EM structure of the RSC complex with the nucleosome, and to show how a particular subunit (Sfh1/BAF47 ortholog) helps facilitate nucleosome ejection (Ye et al., Science 2019).

How is Remodeling and the ATPase ‘motor’ Regulated

Cedric Clapier, Margaret Kasten, Naveen Verma and Tim Mulvihill in my lab are studying how the ATPase subunit is regulated by RSC proteins and histone epitopes to determine when and where nucleosomes are mobilized in the genome.

We have shown that DNA translocation is regulated by proteins that bind the ATPase, and by domains within the ATPase – which determine whether SWI/SNF remodelers like RSC slide nucleosomes, or eject nucleosomes (Clapier et al., Molecular Cell 2016). Of particular interest are two actin-related proteins (ARPs), Arp7 and Arp9 (which we discovered) which bind directly to the ATPase subunit of the RSC remodeler (Szerlong et al., Nature Str. Mol. Biol., 2008) and regulate the nucleosome remodeling reaction by regulating coupling—the efficiency by which each ATP hydrolysis leads to productive DNA translocation (Clapier et al., Molecular Cell, 2016). Importantly, we have shown that fast, efficient, and forceful DNA translocation—promoted by ARPs—is the basis of nucleosome ejection (Clapier et al., Molecular Cell 2020).

We have also shown how RSC sub-complexes are specialized to handle nucleosomes that are fully or only partially wrapped by DNA (Schlichter, Kasten et al., eLife 2020). Additionally, we have examined how the two complexes, RSC and ISWI, antagonize each other in vivo at gene promoters to help regulate nucleosome/chromatin structure and transcription (Parnell, Schlichter et al., eLife 2015). ISWI serves to organize nucleosome arrays and positioning at promoters, whereas the SWI/SNF-family RSC complex provides disorder and access to the promoter, to promote the binding of transcription factors and the act of transcription (Schlichter, Kasten et al., eLife 2020).

Chromatin Remodeler Mutations in Cancer

Mutations in SWI/SNF-family complexes (also called BAF complexes mammals) are found in ~20% of human tumors. These include recessive loss-of-function mutations that prevent SWI/SNF(BAF) targeting or activity. However, we recently found a set of dominant gain-of-function cancer-causing mutations that map to the regulatory ‘hub’ region of the ATPase motor. These mutations all cause the upregulation of DNA translocation and increased nucleosome sliding (Clapier et al., Molecular Cell 2020), suggesting that precocious nucleosome sliding—and linked transcriptional misregulation—likely underlies these cancers.

Another way that SWI/SNF(BAF) complexes are mutated in cancer involves the creation of oncogenic fusion proteins which incorporate into the complex to provide dominant mis-targeting or dysregulation. A prominent example is the SS18-SSX fusion family, which is the cause of synovial sarcoma. The SS18 protein is a member of the BAF complex, but fusion to SSX dysregulates this complex. Recently, we published the mechanism of dysregulation, which involves the oncogenic fusion protein leading to the destruction of particular BAF complexes, while leaving others intact and of even greater proportion—changing the balance of chromatin remodeling in the cell, and impacting chromatin openness and transcription.

Next Steps

We are now exploring how histone modifications/variants and transcription factors influence the activities of these remodelers by interacting with the regulatory ‘hub’. Here, we believe these interactions will be the key to understanding how the ejection and sliding activities are ‘unlocked’ in a targeted/focal manner at particular enhancers and promoters. We have also recently reconstituted the human SWI/SNF(BAF) complex, using a system that now allows us full control over the production of WT, mutant, oncogenic or disease-related remodeling complexes—as well as a system for structural studies. This will provide a versatile and powerful platform for mechanistic studies of human remodelers.