Using a Novel PEV Assay to Identify Heterochromatin Clustering Factors

Olivia working on a computer.

Students

College

Faculty

Assistant Professor of Genetics

Project Summary

This summer, I performed a genetic screen for factors that drive B-compartment formation and/or maintenance in Drosophila melanogaster. This work was supported by the Pincus-Magaziner Family Undergraduate Research and Travel Fund from the College Alumni Society. With these funds, I was able to order over 100 UAS-RNAi D. melanogaster stocks for use in this screen.

My project’s primary goal was to identify novel factors involved in nuclear organization of silent chromatin. The field of three-dimensional nuclear organization sits within the larger field of epigenetics, which is the investigation of gene expression programs that determine cell identity and response to stimuli. Every cell in the human body has the exact same DNA (excluding random somatic mutations), but genes within that DNA are silenced or activated to create distinct cell types with wildly different characteristics. Gene silencing is essential to maintain cell identity, but little is known about how the spatial relationship between silent chromatin contributes to effective silencing. Genome contact maps generated by Hi-C chromatin conformation capture experiments show evidence for the aggregation of silent chromatin into B-compartments1. While work published in 20172,3 suggests that a heterochromatin protein (HP1a) plays an essential role in formation of phase-separated heterochromatin droplets, the formation and maintenance of phase-separated heterochromatin is still largely uninvestigated. 

In this project, I tested putative nuclear organization factors that were identified in a prior screen4. My assay of investigation was a mutant Drosophila strain called brownDominant, which can essentially be described as an induced long-distance interaction between the brown gene and constitutive heterochromatin. By knocking down the level of each candidate protein, I was able to identify factors that influence brown’s interaction with heterochromatin (and by association its silencing) by assessing the amount of pigment produced in the fly eye. I completed my screen the week before the fall semester started and I am especially excited to investigate the role of nucleolar genes in facilitating B-compartment interactions.

In my research experience throughout the spring and summer, I have learned essential time management and organizational skills. While I was already trained in the fly husbandry and immunofluorescent imaging included in this project, the independence that I had in pursuing my scientific questions forced me to think critically about how I could maximize my time and best organize my data. In the future, I look forward to learning how to perform qPCR (quantitative PCR) to measure the degree of heterochromatic silencing following RNAi knockdown of my candidate genes. This project has supplemented my educational experience at Penn by allowing me to pursue my own scientific questions and giving me the time to stay abreast of the new research within my field. My independent research experience has given me the skills that I will need when I pursue a biomedical PhD in epigenetics following my graduation in May 2020.

  1. Lieberman-Aiden E., van Berkum NL, Williams L., Lander ES, & Dekker J (2009)  Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326(5950):289–293. Epub 2009/10/9.
    https://doi.org/10.1126/science.1181369 PMID: 19815776. PubMed Central PMCID: PMC2858594. 
  2. Larson AG, Elnatan D., Keenen MM, Trnka MJ, & Narlikar GJ (2017) Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547(7662):236–240. Epub 2017/6/21. https://doi.org/10.1038/nature22822 PMID: 28636604. PubMed Central PMCID: PMC5606208.             
  3. Strom AR, Emelyanov AV, Mir M., Fyodorov DV, & Karpen GH (2017) Phase separation drives heterochromatin domain formation. Nature 547(7662):241–245. Epub 2017/6/21. https://doi.org/10.1038/nature22989 PMID: 28636597. PubMed Central PMCID: PMC6022742.
  4. Joyce EF, Williams BR, Xie T, & Wu CT (2012) Identification of genes that promote or antagonize somatic homolog pairing using a high-throughput FISH-based screen. PLoS Genetics 8(5): e1002667. Epub 2012/5/10. https://doi.org/10.1371/journal.pgen.1002667 PMID: 22589731. PubMed PMCID: PMC3349724.