Type B histone acetyltransferase Hat1p participates in telomeric silencing

Abstract

Hat1p and Hat2p are the two subunits of a type B histone acetyltransferase from Saccharomyces cerevisiae that acetylates free histone H4 on lysine 12 in vitro. However, the role for these gene products in chromatin function has been unclear, as deletions of the HAT1 and/or HAT2 gene displayed no obvious phenotype. We have now identified a role for Hat1p and Hat2p in telomeric silencing. Telomeric silencing is the transcriptional repression of telomere-proximal genes and is mediated by a special chromatin structure. While there was no change in the level of silencing on a telomeric gene when the HAT1 or HAT2 gene was deleted, a significant silencing defect was observed when hat1Delta or hat2Delta was combined with mutations of the histone H3 NH(2)-terminal tail. Specifically, when at least two lysine residues were changed to arginine in the histone H3 tail, a hat1Delta-dependent telomeric silencing defect was observed. The most dramatic effects were seen when one of the two changes was in lysine 14. In further analysis, we found that a single lysine out of the five in the histone H3 tail was sufficient to mediate silencing. However, K14 was the best at preserving silencing, followed by K23 and then K27; K9 and K18 alone were insufficient. Mutational analysis of the histone H4 tail indicated that the role of Hat1p in telomeric silencing was mediated solely through lysine 12. Thus, in contrast to other histone acetyltransferases, Hat1p activity was required for transcriptional repression rather than gene activation.

FIG. 1

Effects of hat1Δ and histone H3 single-lysine-to-arginine mutations on telomeric silencing. The indicated histone H3 alleles were introduced into UCC1111 (HAT1) and MPY1 (hat1Δ). Telomeric silencing was measured by spotting 10-fold serial dilutions of cells on synthetic complete plates (HC) and synthetic complete plates containing 5-FOA (HC+5-FOA) (Materials and Methods). Plates were photographed after 3 days of growth at 30°C. The relevant genotypes of the strains are indicated on the left. WT, wild type. Assays were performed on at least three separate colonies from each strain.

FIG. 2

Function of Hat1p in telomeric silencing is uncovered by particular histone H3 double-lysine-to-arginine mutations. The indicated histone H3 alleles were introduced into UCC1111 (HAT1) and MPY1 (hat1Δ). Telomeric silencing was assayed as described in the legend to Fig. 1. The genotypes are indicated on the left.

FIG. 3

hat1Δ causes a decrease in the size of 5-FOA-resistant colonies when combined with histone H3 triple-lysine-to-arginine mutations that retain lysine 14. The indicated histone H3 alleles were introduced into UCC1111 (HAT1) and MPY1 (hat1Δ). Telomeric silencing was assayed as in Fig. 1. The genotypes of the strains are indicated.

FIG. 4

Single histone H3 lysine residues can be sufficient to support telomeric silencing. (A) The indicated histone H3 alleles were introduced into UCC1111 (HAT1) and MPY1 (hat1Δ). Telomeric silencing was assayed as in Fig. 1. (B) Analysis of the level of telomeric silencing of histone H3 alleles containing four or five lysine-to-arginine mutations. The indicated histone H3 alleles were introduced into UCC1111. Telomeric silencing was assayed as in Fig. 1. (C) Telomeric silencing was quantitated from three to five repetitions of the assays shown in panels A and B by counting the number of colonies that grew on HC and HC+5-FOA plates. The mean has been plotted, with the error bars representing the standard deviation. Plates were incubated for 7 days to aid in the counting of small colonies.

FIG. 5

HMR silencing is unaffected by hat1Δ/histone H3 mutations. RT-PCRs were performed on total RNA (without removal of genomic DNA) isolated from the MATa strain BY4705a (lane 1) and MATα strains BY4705 (lane 2), UCC6580 (lanes 3 to 8), UCC1111 (lanes 9 and 11), and MPY1 (lanes 10 and 12). UCC1111 and MPY1 also contained the histone H3 alleles indicated. The amount of total RNA in each reaction is indicated. The migration of DNA molecular size standards is given on the left.

FIG. 6

Role of Hat1p in telomeric silencing is mediated through histone H4 lysine 12. The indicated histone H3-H4 alleles were introduced into UCC1111 (HAT1) and MPY1 (hat1Δ). Telomeric silencing was assayed as in Fig. 1.

FIG. 7

Hat2p is involved in telomeric silencing. The indicated histone H3 alleles were introduced into UCC1111 (HAT1), MPY1 (hat1Δ), TKY101 (hat2Δ), and TKY104 (hat1Δhat2Δ). The strains were then assayed for telomeric silencing as in Fig. 1.