Avni Patel – Biomedical Science

Project Title: The Transcriptional Role of PHF20 and its Paralog, PHF20L1, and their Implications in Cancer

Project Mentor: Margarida Santos – Epigenetics and Molecular Carcinogenesis

Introduction: The Non-Specific Lethal (NSL) complex is an important transcriptional regulatory complex that regulates gene expression via facilitating histone acetylation. There are several subunits comprising the NSL complex, two of which are methyl lysine readers: Plant Homeodomain Finger 20 (PHF20) and its paralog, Plant Homeodomain Finger Like 1 (PHF20L1). Little is known about the biochemical and functional properties of these proteins, so this project aims to identify the role of these proteins in transcriptional regulation. To map the genome-wide landscape of these proteins, chromatin Immunoprecipitation (ChIP) was performed on 3x HA-tagged PHF20 and PHF20L1 samples, which were then subjected to next-generation sequencing to identify the binding regions of these proteins. ChIP-sequencing data revealed that PHF20 and PHF20L1 bind primarily to the promoter regions of various target genes; five genes were chosen for further experimentation: NAGPA, PIGT, NUDCD3, VAMP3, and LAMP1. 

Methods: To examine whether PHF20 and PHF20L1 impact target gene expression, Reverse-Transcriptase quantitative PCR (RT-qPCR) was performed after knocking down PHF20 and/or PHF20L1 in vitro using short hairpin RNA (shRNA) technology. A double knockdown in vitro system was generated by sequentially transducing U20S cells with lentiviral vectors containing the sequences for PHF20 and/or PHF20L1 shRNAs. Stable transduction of the desired cell lines was validated via RT-qPCR and Western blotting. RNA was extracted from each cell line, cDNA was synthesized, and RT-qPCR was performed for all five target genes. Target gene expression was assessed relative to housekeeping gene RPLP0.

Results: RT-qPCR and WB data demonstrated acceptable knockdown efficiency levels of the desired proteins in each respective knockdown cell line. Overall, upon knocking down PHF20, PHF20L1, or both proteins, no significant alteration of target gene expression was observed. 

Conclusions: Though these studies do not provide a direct link to the potential uses of these proteins in cancer treatment, they lay a strong foundation for future studies to explore the role of these proteins in both normal and disease states. One area of future interest involves exploring the epigenetic interaction between PHF20 and RAS target genes, as the RAS pathway is often mutated in several cancers.

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