Chromatin-associated genes protect the yeast genome from Ty1 insertional mutagenesis

Abstract

Chromosomal genes modulate Ty retrotransposon movement in the genome of Saccharomyces cerevisiae. We have screened a collection of 4739 deletion mutants to identify those that increase Ty1 mobility (Ty1 restriction genes). Among the 91 identified mutants, 80% encode products involved in nuclear processes such as chromatin structure and function, DNA repair and recombination, and transcription. However, bioinformatic analyses encompassing additional Ty1 and Ty3 screens indicate that 264 unique genes involved in a variety of biological processes affect Ty mobility in yeast. Further characterization of 33 of the mutants identified here show that Ty1 RNA levels increase in 5 mutants and the rest affect mobility post-transcriptionally. RNA and cDNA levels remain unchanged in mutants defective in transcription elongation, including ckb2Delta and elf1Delta, suggesting that Ty1 integration may be more efficient in these strains. Insertion-site preference at the CAN1 locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as BRE1 and RAD6, histone H3 acetylation by RTT109 and ASF1, and transcription elongation by SPT5. Our results indicate that multiple pathways restrict Ty1 mobility and histone modifications may protect coding regions from insertional mutagenesis.

Figure 1.—

Characterization of Ty1 restriction mutants. Ninety-one mutants (1.9%) were identified from 4639 MATα deletion strains, on the basis of an increased level of His⁺ papillation mediated by a chromosomal Ty1 element containing the retrotransposon mobility marker, his3-AI. Thirty-three mutants were chosen for further analyses after considering the function of the deleted gene and the level of Ty1his3-AI mobility, 20 mutants were identified in other screens for Ty1 mobility, and 38 mutants remain to be characterized.

Figure 2.—

Heat map of enrichment scores of GO biological process terms (GOBPs) for gene lists derived from independent screens for Ty1 (Scholes et al. 2001; Griffith et al. 2003) and Ty3 modulators (Irwin et al. 2005). The gradient of red color indicates the enrichment levels with black representing no enrichment [maximum enrichment ≥3 (P-value ≤0.001), no enrichment = 0 (P-value >0.05)]. Rows of the heat map are GOBPs and columns are the genetic screens for Ty modulators. A-R: more detailed GOBPs associated with Ty1 restriction genes identified in this work (supplemental Table S1). A heat map containing all GOBPs associated with Ty1 restriction genes identified here is shown in supplemental Figure S1. B-R: GOBPs associated with Ty1 restriction genes identified by transposon mutagenesis (Scholes et al. 2001). C-H: GOBPs associated with Ty1 helper genes identified by systematic screening of a diploid deletion library (Griffith et al. ;2003). D-R: GOBPs associated with Ty3 restriction genes identified by systematic screening of a haploid deletion library (Irwin et al. 2005). D-H: GOBPs associated with Ty3 helper genes identified by systematic screening of a haploid deletion library (Irwin et al. 2005). Hierarchical clustering of the GOBPs for Ty modulators is shown on the left.

Figure 3.—

Identification of common and unique Ty modulators. Abbreviations are defined in supplemental Figure S1 and in Figure 2. The shaded circle represents Ty1 restriction genes identified in this work (A-R). No genes in common were found when B-R and C-H, and B-R and D-H, were compared. Also refer to Maxwell and Curcio (2007) for further comparative analyses of Ty1 (B-R and C-H) and Ty3 (D-R and D-H) modulators.

Figure 4.—

Frequency estimate of Ty1- and non-Ty1-induced Can^R mutations. Open bars, frequency of Can^R mutations caused by Ty1 insertion; cross-hatched bars, frequency of Can^R mutations caused by other mutational events; solid bars, overall frequency of Can^R mutations. Standard deviations are above the solid bars. On the bottom is the fraction of Can^R mutations caused by Ty1. Also refer to supplemental Tables S4 and S5.

Figure 5.—

Ty1 insertions in the CAN1 promoter region vs. the coding sequence. Shaded bars, Ty1 insertions in the CAN1 promoter region; solid bars, Ty1 insertions in the CAN1 coding sequence. On the bottom are Ty1 restriction genes that were analyzed. P-values were obtained by comparing the distribution of promoter vs. coding sequence insertions in WT and Ty1 restriction mutants (‡, refer to materials and methods). Also refer to supplemental data for more information on the spectrum of Can^R mutations (supplemental Tables S4 and S5), the orientation of the Ty1 insertions (supplemental Table S6), and DNA sequence analysis of Ty1 insertions when PAF1 was deleted (supplemental Figure S5).

Figure 6.—

Relationship between ubiquitination of proteins involved in transcription elongation by RNA polymerase II and by Ty1 transposition. Protein names (in boldface type) required for restricting Ty1 transposition and maintaining target site preference at CAN1 include the Paf complex subunits Cdc73, Paf1, and Rtf1 and the Rad6-Bre1 ubiquitination (+Ub) complex that modifies histone H2B on K123. Rad6-Bre1 may also ubiquitinate additional proteins that have not been identified. The Paf1 complex subunit protein Leo1 is required for restricting Ty1 transposition but is not required for target-site preference at CAN1, and Ctr9 was not analyzed. The Bur1-Bur2 cyclin-dependent protein kinases (+Phos) and the histone H3 methylases (+Me) Dot1 and Set1 were not identified in our screen for Ty1 restriction genes, but may affect Ty1 target preference.