Amplification of histone genes by circular chromosome formation in Saccharomyces cerevisiae - PubMed (original) (raw)
Amplification of histone genes by circular chromosome formation in Saccharomyces cerevisiae
Diana E Libuda et al. Nature. 2006.
Abstract
Proper histone levels are critical for transcription, chromosome segregation, and other chromatin-mediated processes(1-7). In Saccharomyces cerevisiae, the histones H2A and H2B are encoded by two gene pairs, named HTA1-HTB1 and HTA2-HTB2 (ref. 8). Previous studies have demonstrated that when HTA2-HTB2 is deleted, HTA1-HTB1 dosage compensates at the transcriptional level(4,9). Here we show that a different mechanism of dosage compensation, at the level of gene copy number, can occur when HTA1-HTB1 is deleted. In this case, HTA2-HTB2 amplifies via creation of a new, small, circular chromosome. This duplication, which contains 39 kb of chromosome II, includes HTA2-HTB2, the histone H3-H4 locus HHT1-HHF1, a centromere and origins of replication. Formation of the new chromosome occurs by recombination between two Ty1 retrotransposon elements that flank this region. Following meiosis, recombination between these two particular Ty1 elements occurs at a greatly elevated level in hta1-htb1Delta mutants, suggesting that a decreased level of histones H2A and H2B specifically stimulates this amplification of histone genes. Our results demonstrate another mechanism by which histone gene dosage is controlled to maintain genomic integrity.
Conflict of interest statement
The authors declare no competing financial interests.
Figures
Figure 1. Characterization of HTA2-HTB2 amplification in hta1-htb1Δ
a, A simplified genome map of the HTA2-HTB2 region on chromosome II. The white boxes represent the HTA2-HTB2 and HHT1-HHF1 histone loci. The grey boxes represent the Ty1 elements, YBLWTy1-1 and YBLWTy1-2, with the long terminal repeats (known as LTRs or δ elements) shown as black triangles. The grey circle represents the centromere. The probes used in b and c are represented as black bars above the map in a; the probe used for each Southern analysis is indicated below the blots in b and c. A complete map of the region is shown in Supplementary Fig. 1. b, Separated chromosomes from S. cerevisiae strains were analysed on a CHEF gel. Each panel shows a wild-type strain (lane 1), three hta1-htb1Δ strains (lanes 2–4), the hta1Δ strain from the deletion project (lane 5), and an hta2-htb2Δ strain (lane 6). The left panel is an ethidium-bromide-stained gel showing the positions of the chromosomes. The altered migration of chromosome XII is a consequence of the rDNA repeats, which are known to vary in number. The other three panels show analysis of the same gel by Southern hybridization analysis. c, Chromosomes from the same strains were examined as in b using probes just outside (probes A and D) and within (probes B and C) the region flanked by YBLWTy1-1 and YBLWTy1-2. The hta1Δ strain from the deletion set has the identical pattern to our hta1-htb1Δ strains. We assume that during construction of the deletion set, the hta1Δ strain acquired the amplification and the htb1Δ strain did not. Chr., chromosome.
Figure 2. Analysis of newly constructed _hta1-htb1Δ_strains
a, Strains from HTA1-HTB1/hta1-htb1Δ tetrad dissection. Shown is a representative tetrad dissection plate. Each column shows growth of the four progeny from a single tetrad. The large colonies are wild type and the small colonies are hta1-htb1Δ. The apparent slow growth of the hta1-htb1Δ colonies is caused by slow germination (see Supplementary Discussion). Most spores that failed to germinate, for example, tetrad 1 spore D, were also hta1-htb1Δ. b, Four hta1-htb1Δ progeny (lanes 1–4) and two wild-type progeny (lanes 5 and 6) generated from the HTA1-HTB1/(hta1-htb1)Δ tetrad dissection were analysed by a CHEF gel Southern analysis probed with HTA2-HTB2. Controls include the HTA1-HTB1/hta1-htb1Δ parent diploid (lane 7), an hta1-htb1Δ haploid (lane 8) and a wild-type strain (lane 9). All strains were tested with the same probes as Fig. 1 and shown to contain the same amplified region (data not shown).
Figure 3. Tests for the presence of a 39 kb circular chromosome
a, PCR test for recombination between YBLWTy1-1 and YBLWTy1-2. The left side shows an abbreviated view of the HTA2-HTB2 region of chromosome II, before and after recombination between the two Ty1 elements. The arrows indicate the PCR primers designed to amplify a product only if the recombination event occurred. The right side shows an ethidium-bromide-stained gel of the PCR products. Lanes 1–4 are four independently derived hta1-htb1Δ strains containing the amplification. The presence of the 7,500 bp PCR product is consistent with the predicted recombination event. The less intense 930 bp band is probably due to crossover PCR between partly synthesized products. CHA1 is a positive control for the PCR. b, Cleavage of the amplification by HO endonuclease. The left diagram indicates the location of the HO endonuclease site on chromosome II and the amplification. The right side shows an _HTA2-HTB2_-probed CHEF gel Southern analysis of wild-type and hta1-htb1Δ strains, with and without the HO site, before and after HO induction. The 252 kb band in lanes 12 and 16 is a fragment of chromosome II produced by HO cleavage. The 39 kb band in lane 16 indicates a linearized form of the amplification.
Figure 4. Characterization of the recombination event to amplify histone genes
a, Analysis of Ty1 deletion mutants. HTA1-HTB1/hta1-htb1Δ diploids that are homozygous for deletion of either YBLWTy1-1 or YBLWTy1-2 were sporulated and hta1-htb1Δ survivors were analysed for the amplification. Shown is a CHEF gel Southern analysis probed with HTA2-HTB2. YBLWTy1-1Δ hta1-htb1Δ strains (lanes 1–4) and the YBLWTy1-2Δ hta1-htb1Δ strain (lane 5) do not contain the HTA2-HTB2 amplification. Controls are an hta1-htb1Δ mutant with normal Ty1 elements (lane 6) and a wild-type strain (lane 7). b, Replacement of the Ty1 elements. HTA1-HTB1/hta1-htb1Δ diploids in which the Ty1 elements YBLWTy1-1 and YBRWTy1-2 were each replaced with Ylp5, were sporulated and dissected. Surviving hta1-htb1Δ strains (lanes 1–5) and wild-type progeny (lane 6) were analysed as described for a. Controls include the parent diploid (lane 7), an hta1-htb1Δ haploid (lane 8), and a wild-type strain (lane 9). By PCR, the HTA2-HTB2 amplification in the Ty1 replacement hta1-htb1Δ strains was confirmed to be a circular chromosome (data not shown). Note that owing to its slightly smaller size, the band for the Ylp5-containing amplification migrates faster than the Ty1-containing amplification (compare lanes 1–5 with lane 8). c, Frequency of Ty1-Ty1 recombination in wild-type and hta1-htb1Δ strains. The diagram depicts the Ty1 flanked region of chromosome II with the HTA2-HTB2 locus and the Ty1-_URA3_-Ty1 configuration on chromosome IV or XVI. In the Ty1-_URA3_-Ty1 constructs, the Ty1 elements are 17 kb apart, compared with 33 kb for the Ty1 elements on chromosome II. The expected phenotypes of recombinants at both classes of loci are indicated. HTA1-HTB1/hta1-htb1Δ diploid strains were constructed that are homozygous for either Ty1-_URA3_-Ty1 configuration. The diploids were sporulated and analysed for the phenotypes of recombinants at either locus. The table presents the results of the Ty1-Ty1 recombination assay. The amplification frequency was found to be 14–18% in hta1-htb1Δ. In contrast, none of the viable hta1-htb1Δ progeny lost the URA3 marker, demonstrating that the HTA2-HTB2 amplification event occurs at a significantly higher frequency than the Ty1-_URA3_-Ty1 recombination event. All three pairs of Ty1 elements are capable of normal levels of recombination during mitotic growth (Supplementary Table 3). In a separate experiment, the HTA2-HTB2 amplification frequency in wild-type strains following meiosis was found to be close to 0% (see Supplementary Methods).
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