Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization

ABSTRACT Synthetic genome evolution provides a dynamic approach for systematically and straightforwardly exploring evolutionary processes. Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detect over 260 000 rearrangement events after the SCRaMbLEing of a yeast strain harboring 5.5 synthetic yeast chromosomes (synII, synIII, synV, circular synVI, synIXR and synX). Remarkably, we find that the rearrangement events exhibit a specific landscape of frequency. We further reveal that the landscape is shaped by the combined effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. The enormous numbers of rearrangements mediated by SCRaMbLE provide a driving force to potentiate directed genome evolution, and the investigation of the rearrangement landscape offers mechanistic insights into the dynamics of genome evolution.

centromeres of the wild-type chromosome were PCR amplified from the genome of BY4741.

Yeast transformation
Yeast transformations in this study were performed with LiAc/SS/PEG method [8].
Briefly, the overnight YPD saturate culture of yeast cells were diluted into 5 mL fresh

Yeast mating process
Yeast strains of opposite mating types were incubated in selective medium overnight.
Five hundred microliters of each culture were washed with ddH 2 O, and the pellets were resuspended in 3 mL of YPD, and incubated at 30 °C in a shaker incubator at 220 rpm for 8 h. One hundred microliters of the mating suspension were subsequently spread on a selective medium agar plate for single colonies after incubation at 30 °C for 2 days. PCR screening of MATa and MATα was performed to select MATa/α diploid colonies. Forward primer of MATa "ACTCCACTTCAAGTAAGAGTTTG": forward primer of MATα "GCACGGAATATGGGACTACTTCG"; reverse primer of mating type "AGTCACATCAAGATCGTTTATGG".

PCRTag assay for native chromosome elimination
PCRTags are landmarks in synthetic chromosomes including the peritelomeric and pericentromeric regions [9]. The loss of wild-type PCRTags associated with native chromosomes and the retained synthetic PCRTags in synthetic chromosomes were validated by colony PCR to assess the elimination of wtII, III, V, VI, IX, and X, using the primers listed in Supplementary Table 5.
Heterozygous diploid strains were transformed with a pGAL-Vika plasmid with the LEU2 auxotrophic marker [10], and then inoculated into liquid YP+galactose (2%) medium for 12-18 h at 30 °C to turn on the Vika/vox recombination system. Native chromosome II and IX were completely eliminated after Vika recombinase expression was induced. Culture samples of 200 μL were spread on plates with SC-Leu with dextrose medium. After incubation for 2 days at 30 °C, single colonies were replicated on the plates containing YPD+G418 and SC+hygromycin B medium, to identify the colonies that had lost native chromosomes II and IX. The PCRtag assays described above were used to assess the elimination of native chromosomes.
Following the same strategy, wtV, wtX, wtVI, and wtIII were eliminated sequentially, generating a 2n-6 strain. The yYW393 strain was cultured in YPD for 24 h to allow endoreduplication.

Meiosis and sporulation
The yYW393 strain was first transformed with a plasmid containing the MATa and HIS3 markers. Yeasts were incubated in SC-His medium overnight at 30 °C with shaking at 220 rpm, after which 200 µL of the culture was transferred to 3 mL of YPD and then incubated at 30 °C with shaking at 220 rpm for 14 h for early stationary phase culture. The cells were washed with sterile ddH 2 O three times. One milliliter of 50× sporulation medium, 500 µL of the required amino acids (2 g/L uracil, 10 g/L leucine), and 150 µL of 10% yeast extract were mixed together and then diluted with sterile ddH 2 O to a volume of 50 mL. All washed cells were transferred into sporulation solution, mixed well, and subjected to sporulation at room temperature for 5 days followed by dissection. To digest asci, 50 μL cell cultures samples were pelleted by centrifugation (2,000 g × 1 min), re-suspended in 50 μl 0.5 mg/mL 20T zymolyase in 1 M sorbitol at 37 °C for 10 min, and diluted with 300 μl 1 M sorbitol.
Tetrads were dissected under a SINGER Instruments dissection microscope and germinated on YPD plates at 30 °C for 2 days. Nearly 100 tetrads were dismantled to produce one viable haploid. Yeast circular chromosome can cause dicentric chromosome and low spore viability [11,12]. Thus synVI circulation could have been the main cause of observed defective germination. Removal of partial tRNAs may be another reason that affects germination. We also detected 35 mutations including SNPs and Indels in the yYW394 strain. Some of these mutations may have acted as suppressors of defective germination, which is under further investigation in our laboratory.

Adaptive laboratory evolution of yYW394
The yYW394 strain was inoculated in a 5 mL YPD liquid medium with a starting OD600 of 0.1 and grown in a 33 °C shaken incubator at 220 rpm for 24 hours. The cell suspension was then diluted 100-fold into a fresh 5 mL YPD medium, and cultured under the same conditions except that the temperature was set 1 °C higher  Tables S2 and S3.

Preparation of the SCRaMbLEd pool
The SCRaMbLE experiment using pCLB2-Cre-EBD-CYC1t was performed as described previously [13]. Briefly, the yeast strain was transformed with the Probe-Anchor Synthesis (cPAS). Furthermore, to accurately estimate the copy number of target regions, amplification-free sequencing was applied to decrease the likelihood that an appreciable proportion of these sequences would be duplicated and preserve a more even distribution of read coverage across the targeted sequencing regions.

Analyses of novel junctions
Structural variations in the synthetic chromosomes were identified by the alignment of loxPsym sites and neighboring sequences to the yZSJ025 reference genome sequence [14]. Reads containing loxPsym sites and the adjacent sequences of 116 bp on both sides were extracted. The following criteria were used to identify and screen rearrangements for further studies: (1) reads containing the entire 34 bp loxPsym site sequences with flanking sequences belonging to two loxPsym sites of the reference; (2) reads with one end located less than 4 bp from a loxPsym were excluded; and (3) reads containing two or more mismatched bases were excluded.
Identical reads were considered as a result of a single rearrangement event. Only two or more reads supporting a rearrangement event were included in the further analyses.

RNA-Seq Analysis
Yeast strain yZSJ025 was grown from single colonies in liquid YPD culture until reaching early-log phase (OD 600 = 1) at 30 °C with rotation. platform. Peaks in replicates and self-pseudoreplicates were called using MACS2 [18] (--nomodel --extsize 200 --shift -100). Nucleoatac version 0.3.4 was used to call nucleosome positions and occupancy by ATAC data with the default parameters.

Analyses of the ATAC-seq signals
Prior to mapping, standard next-generation sequencing quality control steps using Fragments between 400 and 600 bp were paired-end sequenced on the Illumina Nova-seq PE150 platform.

Construction of the contact map and chromosome 3D model
After quality filtering using Trimmomatic (version 0.38), the clean Hi-C data of two biological replicates for sample yZSJ025, were iteratively mapped to the yZSJ025 genome using the ICE software package (version 1f8815d0cc9e). Dangling ends and other unusable data were filtered, the valid pairs were used to analyze the correlation efficiency of the two biological replicates for each sample using QuASAR-Rep analysis (3DChromatin-ReplicateQC v0.0.1). Then we pooled the data from two replicates together for further analysis. Valid pairs after pooling were binned into 1 kb and 10 kb nonoverlapping genomic intervals to generate contact maps. Raw Hi-C contact maps were normalized using the iterative normalization method to eliminate systematic biases. The chromosomal 3D structure of the strain was inferred using the Pastis (v0.1) method [21]. with a multidimensional scaling (MDS) model. The 10-kb contact maps were used to construct the 3D model.

Statistical analysis
Statistical analysis was performed using R (http://www.r-project.org/) or GraphPad Prism 8.0. Significance was determined via two-tailed, two-sample t tests. Pearson correlation analysis was applied to determine the correlation coefficient (r) and associated p values. A p value of ≤ 0.05 was considered statistically significant.   Table S2 and Table S3. SynVI was tentatively identified as a ring chromosome. Ring chromosomes tend to have a lower sequencing coverage in NGS analysis, which is observable for synVI in yYW393 and yZSJ025.           LoxPsym sites were divided into quintiles according to rearrangement frequency.
Average ATAC-seq signals of each group can be compared among the five groups.
ATAC signals correlate well to the rearrangement frequencies, with the highest ATAC signals observed for Group 1, the lowest for Group 5 and intermediates for Group 2-4.