New mutant versions of yeast FACT subunit Spt16 affect cell integrity

Abstract

Transcription by RNA polymerase II is impeded by the nucleosomal organization of DNA; these negative effects are modulated at several stages of nucleosomal DNA transcription by FACT, a heterodimeric transcription factor. At promoters, FACT facilitates the binding of TATA-binding factor, while during transcription elongation FACT mediates the necessary destabilization of nucleosomes and subsequent restoration of nucleosome structure in the wake of the transcription elongation complex. Altered FACT activity can impair the fidelity of transcription initiation and affect transcription patterns. Using reporter genes we have identified new mutant versions of the Spt16 subunit of yeast FACT with dominant negative effects on the fidelity of transcription initiation. Two of these spt16 mutant alleles also affect cell integrity. Cells relying on these spt16 mutant alleles display sorbitol-remediated temperature sensitivity, altered sensitivity to detergent, and abnormal morphologies, and are further inhibited by the ssd1-d mutation. The overexpression of components of protein kinase C (Pkc1) signaling diminishes this spt16 ssd1-d temperature sensitivity, whereas gene deletions eliminating components of Pkc1 signaling further impair these spt16 mutant cells. Thus, the FACT subunit Spt16 and Pkc1 signaling have an overlapping essential function, with an unexpected role for FACT in the maintenance of cell integrity.

The FACT Histone Chaperone Guides Histone H4 Into Its Nucleosomal Conformation in Saccharomyces cerevisiae

“…When integrated into A364a strains, pob3-T252E caused a moderate Spt 2 phenotype (Lys + after 4 days, whereas pob3-Q308K strains take 2 days), but the HU sensitivity reported previously in a W303 background was not observed ( Figure 8A; a mild defect was reported and we consistently observe weaker phenotypes for FACT mutations in the W303 background relative to A364a due to the different SSD1 alleles in these strains) (O'Donnell et al 2009;and our unpublished observations). However, we did observe the reported suppression of the defects caused by pob3-Q308K, including complete reversal of the Ts 2 and HU phenotypes as well as strong, but not complete, reversal of the Spt 2 phenotype ( Figure 8A).…”

FACT, the Bur Kinase Pathway, and the Histone Co-Repressor HirC Have Overlapping Nucleosome-Related Roles in Yeast Transcription Elongation

“…Impaired transcription-linked nucleosome reassembly can allow aberrant transcription initiation from sequences (‘cryptic promoters’) within the bodies of certain genes. In an earlier study we described the identification and partial characterization of novel mutant alleles of the SPT16 gene that, in dominant fashion, allow effective transcription from cryptic promoters in certain reporter genes [42]. We report here that this dominant phenotype is also caused by several more spt16 mutant alleles (Table 1, which also lists the mutant alleles described previously).…”

“…Moreover, many temperature-sensitive spt16 mutations destabilize the mutant Spt16 protein, making it difficult to distinguish inadequate but normal Spt16 function from mutationally impaired function [40], [41]. To avoid potentially confounding effects due to protein stability, we decided to study the functional relationships of Spt16 by focusing on mutant alleles of the yeast SPT16 gene that affect function in a dominant manner; our reasoning was that a mutant Spt16 protein with a dominant effect would be expected to be relatively stable [42]. The spt16-E857K and spt16-E763G substitution mutations were identified through the impaired control over inappropriate transcription initiation that they cause as a consequence of defective nucleosome reassembly in the wake of transcribing RNAPII: each of these mutations, in dominant (or co-dominant) fashion, activates functional transcription from cryptic promoters within certain reporter genes [42].…”

“…The spt16-E857K and spt16-E763G substitution mutations were identified through the impaired control over inappropriate transcription initiation that they cause as a consequence of defective nucleosome reassembly in the wake of transcribing RNAPII: each of these mutations, in dominant (or co-dominant) fashion, activates functional transcription from cryptic promoters within certain reporter genes [42]. Nonetheless, the Spt16-E857K and Spt16-E763G mutant proteins also supply all essential activities of Spt16 [42]. These observations, coupled with the knowledge that the FACT heterodimer contains a single Spt16 subunit [43], [44], indicates that each mutant version of FACT retains important interactions, but is deficient in interactions that facilitate efficient nucleosome reassembly.…”

Insight Into the Mechanism of Nucleosome Reorganization From Histone Mutants That Suppress Defects in the FACT Histone Chaperone

“…Partial loss-of-function mutations in the genes encoding FACT subunits cause a range of defects in transcription, replication, and other processes (Lycan et al 1994;O'Donnell et al 2004O'Donnell et al , 2009Formosa 2008). Some of these defects can be enhanced by decreasing histone acetylation (Formosa et al 2002), by blocking histone H3-K4 methylation (Biswas et al 2006), by mutating histone genes themselves (Vandemark et al 2008), or by inactivating the Hir/Hpc complex that is involved in regulating histone gene expression and depositing nucleosomes outside of S phase (Formosa et al 2002).…”