Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall A
Hang Phuong Nguyen - Yale University
Co-Author(s): Ivan Surovtsev, Yale University, New Haven, CT; Megan King, Yale University, New Haven, CT.
Diseases such as muscular dystrophy, dilated cardiomyopathy, and cancer are associated with abnormal nuclear shapes and mechanics. It has been demonstrated that chromatin, a complex of DNA and protein that makes up chromosomes, is important to nuclear mechanics and structures. Chromatin’s densely packed region – heterochromatin – is attributed to the binding of Heterochromatin Protein 1 (HP1) to histone H3 methylated on lysine 9 (H3K9me2/3). Heterochromatin is associated with transcriptional gene silencing and with centromeric functions in chromosome segregation. Our group has also found that heterochromatin contributes to the stiffness of the nuclear membrane. Recently, a model for heterochromatin organization based on distinct liquid-like phase has been proposed. Swi6 – an HP1 orthologue in Schizosaccharomyces pombe (S. pombe) – can both undergo liquid–liquid phase separation (LLPS) and compact nucleosomes in vitro [Sanulli, 2018]. However, whether Swi6 undergoes LLPS in vivo and how this contributes to Swi6 functions remains unknown. Our goals are 1) to test if Swi6 displays behavior consistent with LLPS in vivo and 2) to determine if the LLPS behavior of Swi6 contributes to nuclear mechanics. To achieve these goals, it is critical to uncouple the ability of Swi6 to undergo LLPS from its other activities. We hypothesize that swi6-sm1, an allele of Swi6, may be such a separation-of-function allele, as the mutations that comprise the swi6-sm1 allele are in the N-terminal extension, a region of Swi6 implicated in its LLPS activity [Larson, 2017]. While swi6-sm1 cells are defective in gene silencing, they display wild-type-like binding to H3K9me2/3 and supporting of chromosome segregation. Our approach is to use live-cell imaging and fluorescence recovery after photobleaching (FRAP) to quantify and compare the dynamic behavior of wild-type (WT) Swi6-GFP and Swi6-sm1-GFP. Our preliminary data suggest that Swi6-sm1-GFP displays decreased size of heterochromatin region. While further quantitative analysis is needed, thus far, our results are consistent with a role for LLPS in the formation of normal heterochromatin domains. Ultimately, we hope to understand the role of LLPS in chromatin organization and its potential contribution to the integrity of the nuclear envelope.
Funder Acknowledgement(s): This work was funded by a grant from NSF CMMI-1634988 to Megan King.
Faculty Advisor: Megan King PhD, email@example.com
Role: I designed the primers and tagged heterochromatin markers, such as Epe1 and Mst2, with GFP and mCherry. Then, along with my co-authors, I imaged cells that expressed the tagged genes of interest. Finally, I am going to be quantitatively analyze the size, intensity, and motion of the tagged heterochromatin regions with Matlab.