Synthetic chromosome fusion: Effects on mitotic and meiotic genome structure and function

Abstract

We designed and synthesized synI, which is ∼21.6% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. Additional fusion chromosomes were constructed to study nuclear function. ChrIII-I and chrIX-III-I fusion chromosomes have twisted structures, which depend on silencing protein Sir3. As a smaller chromosome, chrI also faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of the crossover-promoting protein Red1. These effects extend over 100 kb and promote disproportionate Red1 enrichment, and thus crossover potential, on small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems.

Keywords: Red1; centromere; chromosome fusion; chromosome splitting; meiosis; synthetic chromosome.

Graphical abstract

Figure 1

The design of synI and construction and characterization of wild-type fusion chromosome strains (A) SynI (180,554 base pairs) encodes all Sc2.0 design features, with a relative length reduction of 21.6% compared to wild-type chrI. Synthetic Universe Telomere Cap replaces the wild-type telomere, and the large deletions at subtelomeres are marked by “X.” CEN1 was removed, allowing synI to be attached to another synthetic chromosome. All retrotransposable elements and introns were deleted. tRNAs were relocated to neo-chromosome. Nineteen TAG stop codons were recoded to TAA, and 62 loxPsym sites were added to the 3′ UTR of non-essential genes and other major landmarks, such as telomeres and sites of tRNA and repeated DNA deletion sites. (B) Schematic outlining the strategy used to construct chrI fusion strains. (C) Schematic showing a CRISPR-Cas9 method deployed to fuse chrI to other chromosomes. (D) The efficiency of the fusion chromosome method for chrI. (E) Pulsed-field gel electrophoresis. Fusion chromosome strain names are indicated atop the gel image. WT, wild-type strain. Red arrows = former location of wild-type chromosomes; blue arrows = new fusion chromosomes. (F) Serial dilution assays to evaluate fitness of fusion chromosome strains. YPD, yeast extract peptone dextrose; SC, synthetic complete medium; YPG, yeast extract peptone with 3% glycerol; YPGE, yeast extract peptone with 3% glycerol and 3% ethanol.

Figure 2

Construction and characterization of synIII-I (A) Schematic showing the strategy to assemble synthetic chromosome I. (B) Pulsed-field gel electrophoresis result. SynIII (∼273 kb, indicated by a black arrow) migrates faster than wild-type chrIII (∼317 kb) and co-migrates with wild-type chrVI (∼270 kb). The attachment of wild-type chromosome I to synIII creates a slower-migrating chromosome (∼496 kb), indicated by a blue arrow. The red arrow indicates the size of the synIII-I fusion chromosome (∼453 kb), which migrates faster than synIII-wtI. (C) Serial dilution growth assay. YPD, yeast extract peptone dextrose; SC, synthetic complete medium; YPG, yeast extract peptone with 3% glycerol. (D) A volcano plot showing RNA-seq data comparing the transcriptome of synIII-I and synIII-wtI strains. Red and blue dots indicate genes whose expression is significantly different in the synIII-I strain compared to synIII-wtI strain. p < 10⁻⁵, |log₂(fold change)| >2.

Figure 3

Construction and characterization of “liberated” synI (A) Schematic illustrating the “telomerase mediated precision splitting” strategy to separate the synthetic chromosome III-I into two synthetic chromosomes III and I. The telomerator contains a URA3 gene with an ACT1 intron, which has an endonuclease I-SceI recognition site (indicated as ∗) flanked by telomere seed sequences (TeSS). (B) PCR results with primers spanning the synIII-I junction and CEN1. The amplicon for junction PCR is 938 bp. The amplicon for wild-type CEN1 is 603 bp, while it is 519 bp in the synIII-I strain with cen1Δ. (C) Pulsed-field gel electrophoresis results. Synthetic chromosomes are marked by triangles, wild-type chromosomes by dots. Blue represents chrIII, and red denotes chrI. SynIII (∼273 kb) comigrates with wild-type chrVI (∼270 kb), while synIII-synI (∼453 kb) comigrates with wild-type chrIX (∼440 kb). (D) Serial dilution growth assay. YPD, yeast extract peptone dextrose.

Figure 4

3D genome organization of wild-type fusion chromosome strains (A) Comparisons of normalized contact maps of fusion chromosome strains and wild-type strains (5 kb bin) and 3D representations inferred from Hi-C contact maps. (B) Violin plots showing the contact frequency of chrI to short (<200 kb), medium (200–400 kb), or long (>400 kb) chromosomal arms, for the wild-type strain (BY4741) and for the strain with chrI fused to the long arm of chrIV. 3D representations are shown in the top of violin plots for visualization comparison; chrI is represented in purple and chrIV in pink; centromeres are colored in red and telomeres in orange.

Figure 5

Twisted structure formed in chrIX-III-I is Sir3 dependent (A) Comparisons of normalized contact maps of wild-type strains and chrIX-III-I strains. Red arrows point at HML and HMR loci. (B) Comparisons of normalized contact maps of chrIX-III-I strains with or without Sir3 protein. Left panel: normalized contact maps. Right panel: ratio between two contact maps (50 kb bin). Blue contacts are stronger in the chrIX-III-I sir3 strain; red contacts are stronger in the chrIX-III-I strain. (C) 3D representations inferred from the contact maps using Shrek 3D. The blank spaces in the junctions on chrIX-III-I sir3 strain reflect the lower mappability of subtelomeric sequences, which were excluded from subsequent analysis.

Figure 6

Distribution of the meiotic axis protein Red1 along fusion chromosomes in heterozygous SK1/S288c hybrid strains (A) Red1 occupancy versus chromosome length in the S288c background. Panels: i, wild type; ii, fusion chromosome IV-I with cen4Δ; iii, fusion chromosome IV-I with cen1Δ; iv, fusion chromosome IX-III-I with cen1Δ and inactive cen3; v, fusion chromosome IX-III-I with cen1Δ and cen9Δ. Chromosomes in fusion chromosomes are indicated by filled circles while wild-type chromosomes are indicated by circles. (B) Red1 occupancy along S288c chr IX for each fusion chromosome strain (colored) overlaid on the wild-type occupancy (black). (C) Mean signal in Red1 peaks along fusion chromosomes as log₂-transformed ratios between each strain and the wild type. Points represent mean peak signal and lines represent local regression (loess normalized).

Figure 7

Meiotic axis protein Red1 occupancy changes upon centromere relocation (A) Mean signal of Red1 peaks along engineered synIV chromosomes as log₂-transformed ratio between inside-out linear and right-side-out linear. In the inside-out linear strains, CEN4 was relocated to the original telomere positions, and the new telomeres are in the original centromere site. Points represent mean peak signal, and lines represent local regression (loess normalized). Note: chromosomal coordinates were shifted by approximately 200 kb for better visualization. The last 10 points from each end were appended to the other end to ensure that the local regression line is continuous across the ends. (B) Red1 occupancy versus chromosome length in the S288c background of engineered synIV strains.