Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi‐C

Abstract In chromosome conformation capture experiments (Hi‐C), the accuracy with which contacts are detected varies due to the uneven distribution of restriction sites along genomes. In addition, repeated sequences or homologous regions remain indistinguishable because of the ambiguities they introduce during the alignment of the sequencing reads. We addressed both limitations by designing and engineering 144 kb of a yeast chromosome with regularly spaced restriction sites (Syn‐HiC design). In the Syn‐HiC region, Hi‐C signal‐to‐noise ratio is enhanced and can be used to measure the shape of an unbiased distribution of contact frequencies, allowing to propose a robust definition of a Hi‐C experiment resolution. The redesigned region is also distinguishable from its native homologous counterpart in an otherwise isogenic diploid strain. As a proof of principle, we tracked homologous chromosomes during meiotic prophase in synchronized and pachytene‐arrested cells and captured important features of their spatial reorganization, such as chromatin restructuration into arrays of Rec8‐delimited loops, centromere declustering, individualization, and pairing. Overall, we illustrate the promises held by redesigning genomic regions to explore complex biological questions.

(2) DpnII, HindIII,SacI,EcoRI,NdeI,SacII,SalI,XbaI,and XhoI. (3) Putative restriction sites are DNA sequences differing with only one base pair from a RS recognized by a RE. (4) The sequence modifications were allowed only in non-forbidden positions. In CDS, silent mutations were introduced. When two sites overlapped, the minimum changes needed were selected. When possible, we favored A ↔ G and C ↔ T substitutions. A validation step to test whether or not the deletion of one site creates a new site was performed after each modification, and if so, a new modification was sought for. (5) Modifications to generate new sites were also only introduced at non-forbidden positions.
Only silent mutations were introduced within coding regions. (6) 583 × 150 kb windows with 10-kb overlaps were generated over the entire genome, excluding telomeres and 75 kb from each side of centromeres. (7) Here, 400, 1,500, 2,000 and 6,000 bp. (8) For each 150-kb window and each interval, the following steps were performed: (i) for each enzyme, for each starting point: putative sites within the first bin of the window (0À 0+spacing). (ii) find the putative sites at position n+1 at a distance interval AE 10% from position n until the end of window.
(9) For each window, a score is calculated as follows: (i) for each interval, a score is calculated for each enzyme based on the median absolute deviation (MAD). (ii) the best enzyme exhibiting the lowest score was chosen for each interval. Each spacing must have a different enzyme, so multiple combinations of enzymes were computed for each window.  coordinates along chromosome 4 (kb) Figure EV2. Restriction pattern (related to Fig 1).
Distribution of the different restriction sites sizes in both the native sequence (top) and Syn-HiC (bottom) 150-kb redesigned sequence.
A Building blocks are iteratively integrated in the genome of S. cerevisiae through homologous recombination following transformation. An alternating auxotrophic selection and counterselection of uracil and leucine are performed to select for transformant likely to have replaced their native sequence by the redesigned one between the two extremities of the targeted region (see Muller et al, 2012 and Annaluru et al, 2014 for details). B Full replacement of the native sequence by the synthetic one is controlled by PCR in transformants cells (SK1 background). For each transformation, PCRTags amplifying either the native or the redesigned genome confirm that the redesigned DNA was integrated over the entire region. The genome of the selected transformant is then confirmed by sequencing. The red lines delimit the region replaced for each transformation (from left to right). C Growth curves of two independent subclones of the selected transformant and two independent subclones of the parent lineage. Each curve is computed out of eight independent cultures. D MA plot representing the differential expression levels for all the genes between the Syn-HiC transformant and the native parental haploid strains. The ORFs within the Syn-HiC redesigned regions whose expression levels are significantly modified are indicated on the graph. Green dots: genes from the Syn-HiC region. Red dots: genes for which the log 10 (P-value) < 0.05.
Source data are available online for this figure.  OD (600) PCT Tag analysis 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 A Normalized contact maps of synchronized populations of cells after 0, 3, 4, and 6 h in sporulation medium as well as of ndt80D-arrested pachytene cells (after 6 h in SPM). The 16 yeast chromosomes are displayed atop the maps. Green arrowheads: inter-centromeric contacts. B Log-ratio of contact maps from (A). The blue-to-red color scale reflects the enrichment in contacts in one population with respect to the other (log2). C Left panels: normalized contact maps of chromosomes V and VI during meiotic time course at t = 4 h (bin size: 5 kb). For chromosome V, a magnification of a~180kb region (dotted box) is displayed under the chromosome. The blue rectangles point at bins enriched in Rec8 protein. Green triangles: centromere position. For chromosome VI, a magnification of a~135-kb region is displayed. Right panel: ratio between the cumulated normalized intra-chromosomal contacts made by 145-kb windows (2.5-kb bins) centered on Rec8-enriched bins or randomly chosen. Blue color shows a depletion of contacts in the random maps, whereas the red signal points at an enrichment in contacts in the maps centered on Rec8-enriched bins.