ABSTRACT
Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.
Subject(s)
Arabidopsis/metabolism , High-Throughput Nucleotide Sequencing/methods , Protein Interaction Mapping/methods , Proteome/metabolism , Transcription Factors/metabolism , Two-Hybrid System Techniques , Arabidopsis/genetics , Proteome/genetics , Sequence Analysis, DNA , Transcription Factors/geneticsABSTRACT
Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.
Subject(s)
Genome, Human , Nuclear Transfer Techniques , Oocytes/metabolism , Polar Bodies/metabolism , Adult , Blastocyst/metabolism , DNA Methylation/genetics , Embryonic Development/genetics , Epigenesis, Genetic , Female , Fertilization in Vitro , Gene Expression Profiling , Genomic Instability , Human Embryonic Stem Cells/metabolism , Humans , Male , Metaphase , Ploidies , Sequence Analysis, RNA , Spermatozoa/metabolism , Spindle Apparatus/metabolism , Transcription, GeneticABSTRACT
Chromosome translocations are well-established hallmarks of cancer cells and often occur at nonrandom sites in the genome. The molecular features that define recurrent chromosome breakpoints are largely unknown. Using a combination of bioinformatics, biochemical analysis, and cell-based assays, we identify here specific histone modifications as facilitators of chromosome breakage and translocations. We show enrichment of several histone modifications over clinically relevant translocation-prone genome regions. Experimental modulation of histone marks sensitizes genome regions to breakage by endonuclease challenge or irradiation and promotes formation of chromosome translocations of endogenous gene loci. Our results demonstrate that histone modifications predispose genome regions to chromosome breakage and translocations.