Project description:Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.
Project description:Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.
Project description:Genome annotation of the chelicerate Tetranychus urticae revealed the absence of many canonical immunity genes. T. urticae either does not mount an immune response or it induces uncharacterized immune pathways. To disentangle these two hypotheses, we performed transcriptomic analysis of mites injected with bacteria vs mites injected with LB-buffer. Two types of bacteria were injected: E. coli and B. megaterium and transcriptomes were sampled 3, 6 and 12 hrs after injection. We found no consistent differential expression after bacterial infection, supporting the hypothesis that spider mites do not mount an immune response. We hypothesize that the apparent absence of inducable immunity pathways in T. urticae is a result of relaxed selective pressure due to ecological factors.
Project description:Rockefeller and Singapore strain Aedes aegypti female mosquitoes differ in the number of bacteria present in the midgut. Females from each strain were either maintained on 3% sucrose solution, fed a sterile blood meal, or fed a blood meal containing a cocktail of bacteria. Differential transcript abundance was compared between females from each strain/treatment combination and a common reference sample pool. The overall goal was to determine how gene expression in Rockefeller females differs from Singapore females in order to better understand why the gut microbiome differs between the strains.
