A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. Reporter constructs, cell culture assays and computational modeling have made major contributions to answering this question, but analysis of elements in their natural context is an important complement. Here, we mutate Notch-dependent LAG-1 binding sites (LBSs) in the endogenous Caenorhabditis elegans sygl-1 gene, which encodes a key stem cell regulator, and analyze the consequences on sygl-1 expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of one LBS in a three-element cluster approximately halved both expression and stem cell pool size, whereas mutation of two LBSs essentially abolished them. Heterozygous LBS mutant clusters provided intermediate values. Our results lead to two major conclusions. First, both LBS number and configuration impact cluster activity: LBSs act additively in trans and synergistically in cis. Second, the SYGL-1 gradient promotes self-renewal above its functional threshold and triggers differentiation below the threshold. Our approach of coupling CRISPR/Cas9 LBS mutations with effects on both molecular and biological readouts establishes a powerful model for in vivo analyses of DNA cis-regulatory elements.
The average person's eyes adapt to darkness within minutes. For those with Oguchi's disease, adaptation can be slowed to several hours. Oguchi disease is an autosomal recessive disorder that results in greatly slowed phototransduction. Phototransduction is a cascade reaction beginning with a photon activating rhodopsin in the rod and leading to hyperpolarization of the cell. Oguchi disease is caused by mutations in rhodopsin kinase which prevent the phosphorylation of rhodopsin, lowering rhodopsin's affinity for arrestin. This reduced ability to bind arrestin decreases the speed in which rhodopsin is deactivated and prepped to reactivate. After a long period in a dark environment, the rhodopsin is eventually deactivated by arrestin, allowing it to be recycled. The Hartford Union High School SMART (Students Modelling a Research Topic) Team has designed a model of rhodopsin kinase to investigate its structure‐function relationship. Oguchi disease can be caused by two different mutations in rhodopsin kinase: large deletion or point mutation. In our 3D model, we will highlight the complete deletion of exon five, the partial deletion at the C‐terminus, and point mutations in the catalytic domain (Val380Asp and Pro391His) that cause Oguchi disease. Understanding the structure‐function relationships of rhodopsin kinase could shed more light on night blindness. This program is supported by a grant from NIH and CTSA.
A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. The use of reporter constructs, cell culture and computational modeling has made enormous contributions to understanding this fundamental question, but analysis of regulatory elements in their natural developmental context is an essential but rarely used complement. Here, we edited Notch-dependent cis-regulatory elements in the endogenous C. elegans sygl-1 gene, which encodes a key stem cell regulator. We then analyzed the in vivo consequences of those mutations – on both gene expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of a single element in a three-element homotypic cluster reduced expression as well as stem cell pool size by about half, while mutation of two elements essentially abolished them. We find that LBS number and LBS neighborhood are both important to activity: elements on separate chromosomes function additively, while elements in the same cluster act synergistically. Our approach of precise CRISPR/Cas9 gene editing coupled with quantitation of both molecular and biological readouts establishes a powerful model for in vivo functional analyses of DNA cis-regulatory elements.Summary statementNotch-dependent DNA cis-regulatory elements work together in their developmental context to shape a transcriptional gradient, control stem cell pool size, and govern differentiation onset.
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