Domain organization of the yeast histone chaperone FACT: the conserved N-terminal domain of FACT subunit Spt16 mediates recovery from replication stress

Abstract

The abundant nuclear complex termed FACT affects several DNA transactions in a chromatin context, including transcription, replication, and repair. Earlier studies of yeast FACT, which indicated the apparent dispensability of conserved sequences at the N terminus of the FACT subunit Cdc68/Spt16, prompted genetic and biochemical studies reported here that suggest the domain organization for Spt16 and the other FACT subunit Pob3, the yeast homolog of the metazoan SSRP1 protein. Our findings suggest that each FACT subunit is a multidomain protein, and that FACT integrity depends on Pob3 interactions with the Spt16 Mid domain. The conserved Spt16 N-terminal domain (NTD) is shown to be without essential function during normal growth, but becomes important under conditions of replication stress. Genetic interactions suggest that some functions carried out by the Spt16 NTD may be partially redundant within FACT.

The FACT Spt16 “peptidase” domain is a histone H3–H4 binding module

“…cerevisiae FACT is an essential nuclear complex involved in transcriptional regulation and chromatin remodeling. Genetic experiments in S. cerevisiae suggest that the Spt16 N-terminal region is dispensable for several FACT functions (21). Similarly, we observe that fission yeast strains expressing ectopically Spt16 mutant proteins that lack the peptidase fold (Spt16-⌬N) are viable (data not shown).…”

“…Indeed, FACT acts as a coactivator of transcriptional initiation and elongation (14), and many Spt16 alleles display genetic interactions with basal transcription factors. Furthermore, FACT subunits biochemically interact with the Pol II elongation complex Paf1 (15), bind the coding region of transcribed Pol II genes, and are recruited to inducible genes upon activation (16-19).FACT's biological roles in transcription (and replication) may stem from its histone chaperone activity (20,21). Histone chaperones stimulate reactions involving the transfer of histones (22), thereby mediating chromatin reorganization.…”

“…FACT's biological roles in transcription (and replication) may stem from its histone chaperone activity (20,21). Histone chaperones stimulate reactions involving the transfer of histones (22), thereby mediating chromatin reorganization.…”

“…Structural information on FACT exists for the HMG-like module Nhp6A from S. cerevisiae (23), which binds DNA, and for the middle domain of S. cerevisiae Pob3 (24), which resembles a double PH-like fold and interacts with replication protein A. The highly conserved Spt16 consists of three domains: an acidic segment at the C terminus (10) that is required for binding histones H2A-H2B in vitro (20) and resembles the acidic domains of Nap1, nucleolin, and Asf1 (25)(26)(27); two central domains, one of which interacts with Pob3 (21,24); and an N-terminal region of Ϸ450 residues (Spt16-N) showing homology with aminopeptidases (28). We were intrigued by the presence of a peptidase-like domain within this essential and conserved histone chaperone [supporting information (SI) Fig.…”

Structure of the Spt16 Middle Domain Reveals Functional Features of the Histone Chaperone FACT

“…Current models suggest that FACT maintains contact with multiple components of nucleosomes simultaneously, preventing dispersal of the nucleosome during transcription (1,7). Consistent with this view, FACT is composed of multiple structural domains that can be detached from one another by limited proteolysis and are well behaved when expressed independently (8,9). The structures of several of these domains have been determined, including the N-terminal domain of Spt16 (Spt16-N) and the N-terminal/ dimerization and middle domains of Pob3 (Pob3-N/D and Pob3-M) (9 -12) (see Fig.…”