ABSTRACT
Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.
ABSTRACT
Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease.
Subject(s)
Protein Aggregates , Saccharomyces cerevisiae Proteins , Superoxide Dismutase-1 , Humans , Amyotrophic Lateral Sclerosis/metabolism , Mutation , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Saccharomyces cerevisiae , Unfolded Protein Response , MeiosisABSTRACT
The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription1-5, but the downstream core promoter in humans has been difficult to understand1-3. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the DPR and the TATA box, as many promoters contain one or the other element. More broadly, these findings show that functional DNA motifs can be identified by machine learning analysis of a comprehensive set of sequence variants.
Subject(s)
Consensus Sequence/genetics , Gene Expression Regulation/genetics , Promoter Regions, Genetic/genetics , RNA Polymerase II/metabolism , Support Vector Machine , Transcription, Genetic , Base Sequence , Cells/metabolism , Computer Simulation , Datasets as Topic , HeLa Cells , High-Throughput Nucleotide Sequencing , Humans , Models, Genetic , Mutagenesis , TATA Box/geneticsABSTRACT
El objetivo de este estudio fue determinar la evolución del glaucoma congénito primario en niños menores de 3 años atendidos en el Centro Nacional de Oftalmología Dr. Emilio Álvarez Montalbán, en el período de enero 2004 a diciembre 2008. El diseño del estudio fué descriptivo de corte transversal. La muestra la conformaron 20 pacientes, constituyendo 57.1% del universo, para un total de 24 ojos. El tipo de muestra fue no probabilística por conveniencia. Los resultados más importantes fueron que el 75% de los niños eran menores de 1 año, encontrándose en el grupo de 4-6 meses un 35%. El sexo masculino constituyó el 70%. Más de la mitad de los pacientes tenían procedencia fuera de Managua. El 80% de los pacientes tenían afectación unilateral. El lagrimeo constituyó el síntoma principal en más de la mitad de los pacientes, la excavación papilar en más del 50% de los ojos era mayor de 0.7, el diámetro corneal promedio fué de 13.6mm, la tonometría inicial promedio fué de 2.3/ 5.5 grs. y la tonometría final promedio fué de 6.5/5.5 grs., cerca de la mitad de los pacientes usaron tratamiento antiglaucomatoso en el prequirúrgico y el 70.8% de los pacientes tenían defecto refractivo miópico. En más del 90% de los ojos se realizó trabeculectomía con un antimetabolito.Se concluyó que la cirugía que se realizó en el 79.2% de los ojos de los pacientes fué trabeculectomía con 5 fluorouracilo, con lo cual se obtuvo un control de la PIO en el 62.5% ojos, reducción del edema corneal en 37.5% y la excavación papilar en el 12.5%.No hubo cambios en el diámetro corneal.Se recomendó elaborar un protocolo de manejo del glaucoma congénito y gestionar el equipo e instrumental necesario para la realización de goniotomía y trabeculotomía que son las cirugías de primera elección en el glaucoma congénito...
