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ABSTRACT: Sequencing S. cerevisiae with synthetic chromosome XI

PROVIDER: PRJNA1007585 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25706730_1.fastq.gz	Fastqsanger.gz
	SRR25706731_1.fastq.gz	Fastqsanger.gz

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Project description:We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.

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Expression data from the partially synthetic yeasts SynIXR-1D, -6B, -22D, and controls.

Project description:We have replaced the right arm of chromosome IX in Saccharomyces cerevisiae with a synthetic version to generate synIXR haploids. The synthetic chromosome features multiple sequeunce modifications. We analyzed gene expression by microarray analysis in three synIXR haploids (1D, 6B, and 22D) to detect any changes in synIXR transcripts or global compensatory changes.

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Hbo1 and Msl complexes preserve differential compaction and H3K27me3 marking of active and inactive X chromosomes during mitosis

Project description:In mammals, chromosome-wide regulatory mechanisms ensure a balance of X-linked gene dosage between males (XY) and females (XX). In female cells, expression of genes from one of the two X-chromosomes is curtailed, with selective accumulation of Xist-RNA, Xist-associated proteins, specific histone modifications (eg. H3K27me3) and Barr body formation observed throughout interphase. Using chromosome flow-sorting, we show that during mitosis, Xist-associated proteins dissociate from inactive X (Xi) chromosomes, while high levels of H3K27me3 and increased compaction of the Xi relative to active X (Xa), are retained. Proteomic comparison of mitotic Xi and Xa revealed, unexpectedly, that components of Hbo1 and Msl/Mof histone acetyltransferase complexes co-enrich with Xa, while inhibitors of histone acetylation co-enrich with Xi. Furthermore, inhibition of Hbo1 or deletion of Msl/Mof components functionally abolishes mitotic differences in H3K27me3 marking and chromosome compaction. These data uncover critical roles for acetylation pathways in preserving X chromosome properties during mitosis.

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