Ashleigh Purvis
Yale University
Identifying direct transcriptional targets of SOX10 using HaloPROTAC mediated degradation in primary patient melanoma
In melanoma, SRY-box transcription factor 10 (SOX10) plays a critical role in disease progression through involvement in cell growth and proliferation pathways. The exact mechanism by which SOX10 is involved in these pathways is poorly understood due to cell viability limitations in traditional gene editing and silencing approaches. We have utilized HaloPROTAC mediated degradation in combination with an inducible CRISPR/Cas9 system to study the transcriptional effects of SOX10 while minimizing genetic compensation. By employing the use of a HaloTag-SOX10 construct to compensate for cell growth and proliferation, endogenous SOX10 can be knocked out without compromising cell viability. After treatment with HaloPROTAC3 to degrade HaloTag-SOX10, changes on the transcript level can be assessed using various sequencing experiments such as RNA-seq, ATAC-seq, and CUT&RUN. The minimal genetic compensation and enhanced temporal control of this approach allows for a novel set of direct transcriptional targets to be identified. New direct gene targets can provide informative insight into the mechanistic role SOX10 plays in proliferative and migratory pathways in melanoma.
In melanoma, SRY-box transcription factor 10 (SOX10) plays a critical role in disease progression through involvement in cell growth and proliferation pathways. The exact mechanism by which SOX10 is involved in these pathways is poorly understood due to cell viability limitations in traditional gene editing and silencing approaches. We have utilized HaloPROTAC mediated degradation in combination with an inducible CRISPR/Cas9 system to study the transcriptional effects of SOX10 while minimizing genetic compensation. By employing the use of a HaloTag-SOX10 construct to compensate for cell growth and proliferation, endogenous SOX10 can be knocked out without compromising cell viability. After treatment with HaloPROTAC3 to degrade HaloTag-SOX10, changes on the transcript level can be assessed using various sequencing experiments such as RNA-seq, ATAC-seq, and CUT&RUN. The minimal genetic compensation and enhanced temporal control of this approach allows for a novel set of direct transcriptional targets to be identified. New direct gene targets can provide informative insight into the mechanistic role SOX10 plays in proliferative and migratory pathways in melanoma.
