Mutant Versions of the S. cerevisiae Transcription Elongation Factor Spt16 Define Regions of Spt16 That Functionally Interact with Histone H3

Abstract

In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16 – the Spt16-M domain – is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation.

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

“…Relevant questions include: do the dimers make bidentate interactions with the same (H3-H4) 2 tetramer while it is in a canonical nucleosome structure, do they promote disassembly of the tetramer from other nucleosomal components, or do they promote disassembly of the (H3-H4) 2 tetramer into dimers? The locations of residues identified as important in various genetic screens (15,16,18,29) are consistent with the conclusion that Spt16-M surfaces participate in extensive interactions, including those relevant for the functional interaction with H3-H4. Beyond the H3-H4 interactions, it will also be important to understand how Spt16-Pob3 interacts with other nucleosomal components.…”

“…The L61W mutation in histone H3 disturbs the structure of nucleosomes, presumably by destabilizing the interface between H3 and H4 (29). Growth defects caused by this mutation can be suppressed by mutations in SPT16 (15,29) that map to Spt16-M. Although the suppression mechanism is not known, these observations indicate a functional link between Spt16-M and histones H3-H4.…”

“…For this mutant in particular, it is tempting to speculate that the phenotype results from disruption of a functional interaction because it is close to a loop that has been implicated in H3-H4 (18)). Glu-857 (blue) was identified in both studies, mutated to either a Gln (15), or a Lys (18). E and F, F o Ϫ F c omit map contoured at 3ϫ r.m.s.d.…”

“…Genetic studies have indicated that the Spt16-M domain has key roles in the physiological function of FACT because many of the mutations that affect FACT function map to this domain (15,16). Mutations in Spt16-M can be suppressed by altering histones to destabilize the interface between H2A-H2B dimers and H3-H4, and mutations in H3 can be suppressed by mutations in Spt16-M (15,17,18).…”

“…Mutations in Spt16-M can be suppressed by altering histones to destabilize the interface between H2A-H2B dimers and H3-H4, and mutations in H3 can be suppressed by mutations in Spt16-M (15,17,18). Spt16-M therefore appears to make important contacts with histones during nucleosome reorganization by FACT, but more detailed mechanistic insights have been unavailable due to the lack of structural information.…”