Journal: The EMBO Journal
Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner
Figure Lengend Snippet: Heterologous activation of the IL8 enhancer triggers IL8 and CXCL1 expression A CRISPR activation (CRISPRa) strategy was applied to test enhancer functions individually. This approach involves a “dead” Cas9 ( blue ) and VP64 ( green ) fusion protein that recruits the NF‐κB ( orange ) and HSF1 ( red ) transactivation domains via MS2 recognition of two stem loops in the sgRNA scaffold ( magenta ). These complexes were targeted to the IL8 or CXCL2 enhancers and promoters (highlights) via different sgRNA pools in HeLa. The position of individual sgRNAs used for CRISPRa is shown in more detail in Fig EV1 . Left bar graphs: Wild‐type HeLa cells were transiently transfected with different combinations of plasmids encoding the “dead” Cas9‐VP64 fusion protein, the MS2‐p65‐HSF1 fusion protein, and empty sgRNA vector or versions containing sgRNAs targeting the IL8 enhancer or promoter. Twenty‐four hours post‐transfection, cells were lysed and total RNA was analyzed for expression changes of the indicated genes compared to samples carrying dCas9‐VP64 and MS2‐p65‐HSF1 fusions, but no sgRNAs. Right bar graphs: The same experiments were performed using sgRNAs targeting the CXCL2 enhancer and promoter. Data information: All data are from four independent transfections. Shown are mean values ± SEM. P ‐values are derived from unpaired t ‐tests comparing every condition against cells expressing all transactivators but lacking sgRNAs (first lane in each graph). Only significant differences are marked by asterisks.
Article Snippet: Plasmids and transient or stable transfections The following plasmids were gifts or were obtained commercially: pSpCas9(BB)‐2A‐Puro (pX459; Addgene (#48139)), pSpCas9(BB)‐2A‐Puro V2.0 (pX459V2.0, Addgene (#62988)), lenti‐sgRNA(MS2)‐zeo backbone (Addgene, #61427), lenti dCAS9‐VP64_Blast (Addgene, #61425), and lenti MS2‐P65‐HSF1_Hygro (Addgene, #61426).
Techniques: Activation Assay, Expressing, CRISPR, Transfection, Plasmid Preparation, Derivative Assay