Yeast Ty1 retrotransposition is stimulated by a synergistic interaction between mutations in chromatin assembly factor I and histone regulatory proteins

Abstract

A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition. The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutaions of cac3 and hir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition. We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA. Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3 hir3 double mutant. In addition, the growth rate of the double mutant was reduced. HIR3 belongs to a class of HIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I. Other combinations of mutations in cac and hir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate. A model explaining the synergistic interaction between cac and hir mutations in terms of alterations in chromatin structure is proposed.

FIG. 1

Ty1 transposition assay. The most common way for the His⁻ strains containing the promoterless his3-Δ4 allele diagrammed above to revert to His⁺ is by the insertion of an upstream Ty1 element. Such insertions were detected by the appearance of 400- to 500-bp PCR amplicons when the HIS3 (1His3) and delta element (U5-in) primers were used. No insertions were detected when the HIS3 (1His3) and delta element (U3-in) primers were used, indicating that the Ty1 elements had to be in the indicated orientation to cause a His⁺ reversion. Arrows in boxes indicate the direction of transcription.

FIG. 2

Characterization of the htr1 mutant. (A) Complementation of htr1 MMS sensitivity. Transformants of L1356 (wild type [WT]) and L1561 (htr1) with the indicated plasmids (YCp50, pCAC3, and pHIR3) were spotted from −Ura onto YPD and YPD plus 0.02% MMS. (B) Localization of htr1-complementing activity. Restriction maps of the cloned fragments in plasmids pCAC3 (top) and pHIR3 (bottom) and subcloned DNA fragments described in the text are shown. +, MMS sensitivity was complemented by the fragment; −, no complementation occurred. Arrows indicate the direction of transcription. Restriction sites are abbreviated as follows: B, BamHI; Bg, BglII; Bs, BssHII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; S, SalI; Sn, SnaBI; Sp, SphI; P, PstI; X, XhoI; and Xb, XbaI. (C) Deletion of HIR3 does not affect levels of Ty1 mRNA. RNA blot analysis of Ty1 (and Ty2) versus TEF1 (internal control) mRNAs from L1356 and the isogenic hir3::LEU2 deletion strain, L1675.

FIG. 3

The htr1 mutation depressed transcription of HTA1-HTB1 and failed to complement hir3. RNA blot analysis was performed after 30 min of growth in the absence (−) or presence (+) of HU. The diploids were formed by crossing SL1006-1B (htr1) and SL1006-1D (HTR1) with W303 (HIR3) and W303Δ3 (hir3). The constitutively transcribed gene PRT1 was used as an internal loading control since its abundance is not affected by HU.

FIG. 4

Double mutations of hir and cac cause MMS sensitivity. (A) Deletion of HIR3 causes MMS sensitivity in the presence of cac3 or cac2. L1356 was found to contain a relevant mutation, cac3-1. L1356 (cac3 HIR3) was transformed with YCp50 or pCAC3, and L1675 (cac3 hir3) was transformed with YCp50, pCAC3, or pHIR3. Cells were grown on −Ura medium and were then spotted onto YPD and YPD plus 0.025% MMS. UV irradiation was at 90 J/m². (B) Deletion of HIR3, HIR1, or HIR2 in L1356 causes MMS sensitivity. MMS sensitivity was scored by spotting 10-fold serial dilutions of cells on YPD with 0.02% MMS (incubation at 30°C for 2 days).

FIG. 5

Silencing and growth defects in hir3 cac3 double-deletion mutants. Strain UCC4543 (wild type [WT]) and its deletion derivatives L1678 (cac3), L1687 (hir3), and L1688 (cac3 hir3) were used. (A) Silencing of telomere VII-L-located SUP4-o (SUP4-o-TEL) is released in hir3 cac3 double-deletion mutants. Expression of SUP4-o-TEL was assayed by spotting 10-fold serial dilutions on SC (control) and −Ade and incubating the plates at 30°C for 4 days. The slight increase in growth of the hir3 strain shown on −Ade was not reproducible. (B) Deletion of both HIR3 and CAC3 retards cell growth. The indicated strains were diluted, plated on YPD, and incubated at 20°C.

FIG. 6

Nuclear localization of HA-tagged Hir3p. (A) Immunogold labeling of HA-Hir3p in L1675. (B) Indirect immunofluorescence and 4′,6-diamidino-2-phenylindole (DAPI) staining of HA-Hir3p in YJB2306.