Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
The 3D architecture of the genome governs its maintenance, expression and transmission. The cohesin complex organises the genome by topologically linking distant loci and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres 1 – 6 . Here we report the 3D structure of budding yeast pericentromeres and establish the relationship between genome organisation and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organised into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Re-orienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation in mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded and the re-structuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between 3D genome organization of a specific chromosomal domain and cellular function.
Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.
BackgroundGlioma stem cells (GSCs) are a subpopulation of stem-like cells that contribute to glioblastoma (GBM) aggressiveness, recurrence, and resistance to radiation and chemotherapy. Therapeutically targeting the GSC population may improve patient survival, but unique vulnerabilities need to be identified.ResultsWe isolate GSCs from well-characterized GBM patient-derived xenografts (PDX), characterize their stemness properties using immunofluorescence staining, profile their epigenome including 5mC, 5hmC, 5fC/5caC, and two enhancer marks, and define their transcriptome. Fetal brain-derived neural stem/progenitor cells are used as a comparison to define potential unique and common molecular features between these different brain-derived cells with stem properties. Our integrative study reveals that abnormal expression of ten-eleven-translocation (TET) family members correlates with global levels of 5mC and 5fC/5caC and may be responsible for the distinct levels of these marks between glioma and neural stem cells. Heterogenous transcriptome and epigenome signatures among GSCs converge on several genes and pathways, including DNA damage response and cell proliferation, which are highly correlated with TET expression. Distinct enhancer landscapes are also strongly associated with differential gene regulation between glioma and neural stem cells; they exhibit unique co-localization patterns with DNA epigenetic mark switching events. Upon differentiation, glioma and neural stem cells exhibit distinct responses with regard to TET expression and DNA mark changes in the genome and GSCs fail to properly remodel their epigenome.ConclusionsOur integrative epigenomic and transcriptomic characterization reveals fundamentally distinct yet potentially targetable biologic features of GSCs that result from their distinct epigenomic landscapes.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1420-6) contains supplementary material, which is available to authorized users.
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