3 research outputs found
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Analysis of Transcription Activation Distance as a Polygenic Trait in Saccharomyces cerevisiae
Much of the eukaryotic transcriptional machinery is conserved from yeast to human. However, the distance over which transcriptional activation can occur differs between Saccharomyces cerevisiae and metazoans. In S. cerevisiae, the upstream activating sequence (UAS) is generally found within 300 base pairs of the transcription start site; when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as 100 kilobases from the start site. In past work, our lab identified five genes that, when mutant, allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As this long-distance activation phenotype was weak, we have now studied long-distance activation as a polygenic trait, isolating strains with multiple mutations that together confer a strong phenotype. To do this, we constructed strains containing two reporters, HIS3 and URA3. For each reporter, the GAL1 UAS was placed approximately 800 base pairs upstream of the transcription start sites. By iterative selection for stronger and stronger expression of HIS3, followed by screening for stronger expression of URA3, we isolated three strains, each containing multiple mutations that contribute to the strength of the long distance activation phenotype. Causative mutations were identified in MOT3, GRR1, MIT1, PTR3, YOR019W, and MSN2 that contribute to the long distance activation phenotype. Strains containing multiple mutations were found to activate the reporter construct at distances up to 2 kilobases. Microarray analysis revealed genome wide transcriptional changes in the mutant strains. Statistical analysis of the microarray results suggests other potential sites of long distance activation throughout out the genome. These results have extended our understanding of mutations that allow long distance activation and have demonstrated the value of studying a phenotype as a polygenic trait
Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.
We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities
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Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae
T Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to
human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant
phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance