Abstract
A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.
Highlights
In eukaryotes, epigenetic information is encoded in chromatin and used for genome regulation and stability [1]
SSRP1 and Spt16 are multi-domain proteins, In animal and plants, FACT is a heterodimer formed by two subunits: a suppressor of Ty 16 and both of them contain dimerization domains (DD), middle domains (MD), and acidic C-terminal (Spt16) and a structure-specific recognition protein 1 (SSRP1)
Activities of three protein complexes were compared in the present study: a complex without the NTD of Spt16, FACT-N without the C-terminal domain (CTD) and high mobility group (HMG) domains of SSRP1 (FACT-N(SSRP∆)) and FACT-N without both acidic domains
Summary
Epigenetic information is encoded in chromatin and used for genome regulation and stability [1]. FACT plays a role in nucleosome survival during transcript elongation by RNA polymerase II, chromatin structure or which, in H2A-H2B turn, prevents transcription initiation from cryptic promoters [5,6,7]. SSRP1 and Spt are multi-domain proteins, In animal and plants, FACT is a heterodimer formed by two subunits: a suppressor of Ty 16 and both of them contain dimerization domains (DD), middle domains (MD), and acidic C-terminal (Spt16) and a structure-specific recognition protein 1 (SSRP1). ANP32E (acidic nuclear phosphoprotein kDa) binds a domain and this interaction is important for H2A.Z deposition on promoter 32 regions. Histone variant, H2A.Z, through its acidic domain and this interaction is important for H2A.Z. CTD of Spt is involved in H2A-H2B binding by FACT [16] and participates in nucleosome deposition on promoter regions.
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