The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming.

The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit's AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers.

ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3-H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A-H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A-H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z-H2B dimers out of nucleosomes and replacement by H2A-H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.

Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.

Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes.

The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1.

Dynamic regulation of transcription factors by nucleosome remodeling.

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.