Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization

Abstract

The essential histone chaperone FACT (cilitates hromatinranscription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization , indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.

Transcription promotes the interaction of the FAcilitates Chromatin Transactions (FACT) complex with nucleosomes inS. cerevisiae

“…While, the altered nuclease sensitivity of Spt16-bound nucleosomes is consistent with a preference of FACT for transcription-disrupted nucleosomes, FACT is also reported to alter nucleosome structure in vitro (M c C ullough et al, 2018; X in et al, 2009), and thus a modified nucleosome structure could also be a consequence of FACT binding. To differentiate between these two possibilities, we rationalized that, if FACT modifies nucleosome structure on its own, then the altered pattern of digestion should be independent of the destabilizing stress of transcription.…”

The histone chaperone FACT modulates nucleosome structure by tethering its components

“…Human FACT (hFACT) is a heterodimer composed of Spt16 and SSRP1 (Orphanides et al, 1999). Yeast FACT additionally requires Nhp6, an HMG-B domain subunit that in metazoans is fused to SSRP1 (McCullough et al, 2018). The FACT complex interacts with all three RNA polymerases (Birch et al, 2009, Tessarz et al, 2014 and facilitates transcription by disrupting nucleosomes in their path, and by aiding in the re-deposition of histones posttranscription (Belotserkovskaya et al, 2003, Formosa et al, 2002.…”

Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in Saccharomyces cerevisiae

“…FACT is a heterodimer of Spt16 with either Pob3 (in yeast and fungi) or SSRP1 (in higher eukaryotes), with each subunit comprising multiple histone-binding modules connected by unstructured linkers (Winkler and Luger 2011;Kemble et al 2015;Tsunaka et al 2016). SSRP1 has an intrinsic DNA-binding domain not found in Pob3, but both versions of FACT require additional free-standing DNA-binding activity to support full activity (Valieva et al 2016;McCullough et al 2018). The High Mobility Group B (HMGB) family member Nhp6 provides this function in yeast (Stillman 2010;Valieva et al 2016;McCullough et al 2018).…”

“…SSRP1 has an intrinsic DNA-binding domain not found in Pob3, but both versions of FACT require additional free-standing DNA-binding activity to support full activity (Valieva et al 2016;McCullough et al 2018). The High Mobility Group B (HMGB) family member Nhp6 provides this function in yeast (Stillman 2010;Valieva et al 2016;McCullough et al 2018). FACT is proposed to use these domains to bind multiple sites on nucleosomes, sequentially exposing and engaging additional buried sites to ultimately produce an altered structure, which we have called the "reorganized" nucleosome (Winkler and Luger 2011;Formosa 2012;Hondele and Ladurner 2013;Tsunaka et al 2016;McCullough et al 2018).…”

“…The High Mobility Group B (HMGB) family member Nhp6 provides this function in yeast (Stillman 2010;Valieva et al 2016;McCullough et al 2018). FACT is proposed to use these domains to bind multiple sites on nucleosomes, sequentially exposing and engaging additional buried sites to ultimately produce an altered structure, which we have called the "reorganized" nucleosome (Winkler and Luger 2011;Formosa 2012;Hondele and Ladurner 2013;Tsunaka et al 2016;McCullough et al 2018). The nucleosome assembly activity of FACT (Belotserkovskaya et al 2003) is then proposed to involve reversal of these steps.…”