Proximity dependent biotin-tagging to screen for proteins involved in FMR1 gene silencing in Fragile X syndrome

Authors

• HT Van
• JE Lee
• YK Park
• K Ge

Abstract

Repeat Expansion Diseases (REDs) are a group of more than 40 human diseases caused by expansion of a disease-specific short tandem repeat tract within a gene. Fragile X syndrome (FXS), the most common heritable form of intellectual disability, is caused by the expansion of a CGG-repeat tract in the 5' UTR of the FMR1 gene. FMR1 alleles with >200 repeats are hypermethylated and transcriptionally silenced. Repeat-induced gene silencing is also seen in other REDs; however, the underlying mechanism is not completely understood. To identify proteins involved in FXS gene silencing, I am developing an unbiased proximity-labeling screen to identify proteins that associate with FMR1 in stem cells derived from unaffected and affected individuals. I have generated FXS embryonic stem cell lines expressing doxycycline-inducible dCas9-APEX2 and repeat-specific CRISPR guide RNA. I will use Chromatin Immunoprecipitation to confirm targeting of dCas9-APEX2 to the FMR1 locus. Addition of doxycycline, together with biotin-phenol and H2O2, will biotin-tag proteins within a ~20 nm radius of the APEX2 peroxidase. Biotinylated proteins will then be purified by streptavidin pull-down and identified by mass spectrometry. Additional cell lines will be produced to assess the proteomic landscape at FMR1 when the gene is experimentally reactivated and in cells with repeat numbers below the silencing threshold. Comparison of these different proteomes should allow proteins contributing to the initiation and maintenance of silencing to be identified. The results obtained from this study may contribute to the understanding of repeat-induced gene silencing in FXS and other REDs.

Scientific Focus Area: Molecular Biology and Biochemistry

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