Using dCas9 to attenuate transcription and its effect on active promoters in Drosophila melanogaster

STEFANIE, BRIZUELA; EZEQUIEL, NAZER; LEI, ELISSA P

Simposio; Summer Poster Day; 2018

NIH

In Drosophila melanogaster, AGO2 chromatin associationdepends on active transcription. Previous studies using ChIRPseq(chromatin isolation by RNA purification) and ChIP-seq(chromatin immunoprecipitation) has suggested AGO2 isrecruited to active promoters co-transcriptionally and thentransferred to nascent mRNA. To elucidate the molecularmechanism driving AGO2 chromatin association and how itattenuates transcription, Kc hemocyte cells were treated with theuniversal transcription initiation inhibitor, Triptolide. Using ChIPseq,untreated cells exhibited AGO2 association with activepromoters while Triptolide treated cells revealed a strong loss ofAGO2 at the transcription start site (TSS). Because Triptolideinhibits the initiation phase of transcription, this project focuseson inhibiting the elongation phase at the promoter region usingdCas9 technology. The objective is to observe if inhibitingdifferent stages of transcription impairs AGO2?s ability toassociate at active promoters when stalling RNA polymerase II.dCas9 (dead CRISPR associated protein 9) is a version of Cas9where endonuclease activity, responsible for modifying DNA, isremoved via point mutations. This allows for the mediation ofgene regulation through an RNA-guided manner using its singleguide RNA (sgRNA). Two sgRNAs were designed downstream ofthe TSS with respect to the candidate genes, betaTub60D andCV2, to inhibit transcription at the elongation phase. A westernblot analysis was used to verify if transfected Kc cells wereexpressing dCas9. After verifying the success of dCas9expression, quantitative RT-PCR (reverse transcriptasepolymerasechain reaction) was used to observe mRNA levels.ChIP quantitative PCR was used to detect the dCas9 and AGO2chromatin association in both candidate genes. Our findingsshow that transcription was not attenuated, AGO2 associationwas not observed at promoters of both candidate genes, andtethering of dCas9 downstream of the TSS was not successful.