Poster abstracts
Poster number 163 submitted by Genine Collins
Inducing HSAT2 Expression in Primary Fibroblast Cells
Genine Collins (Biology Department, Swarthmore College), Christina Rabeler (Biology Department, Swarthmore College), Anusha Bhatia (Biology Department, Swarthmore College), Dawn Carone (Biology Department, Swarthmore College)
Abstract:
Human Satellite 2 (HSAT2) is a noncoding sequence of satellite DNA that is composed of 26 base pair tandem repeats and is located near the centromere of many chromosomes. In cancer cells, heterochromatin decondensation enables transcription of HSAT2, resulting in nuclear RNA accumulations that sequester RNA binding proteins such as TDP-43 and contribute to physical cell defects (Hall et al. 2017). Despite its repetitive nature, HSAT2 is intrinsically heterogeneous and locations on chromosome 7 and 17 are most highly expressed in U2OS osteosarcoma cells (Rabeler et al. 2024). We hypothesize that the propensity for HSAT2 RNA to form these accumulations and recruit proteins is due to locus specific sequence differences rather than chromosomal location.
We previously conducted sequence analysis on several heterogeneous locations of HSAT2 in the genome, and identified highly and lowly expressed variants on chromosome 7. The forward and reverse complement of each location (as well as a scramble control with the same A/T richness as HSAT2) were cloned into the inducible AAVSI-TRE3G TALEN plasmid.
To study HSAT2 expression, we transfected the recombinant plasmids into WI-38 primary fibroblast cells at the AAVS1 safe harbor locus on chromosome 19. Previous attempts to do so, via a lipid-mediated delivery system, had limited transfection efficiencies and were ineffective in establishing stable lines. By optimizing an electroporation procedure, we achieved a higher transfection efficiency and established stable lines.
By inducing similar expression levels across all inserted sequences and monitoring the stable lines over fixed intervals, we can study the rate at which nuclear accumulations form, analyze the size and intensity of these accumulations, and compare TDP-43 distribution across each condition.
References:
Hall LL, Byron M, Carone DM, Whitfield TW, Pouliot GP, Fischer A, Jones P, Lawrence JB. Demethylated HSATII DNA and HSATII RNA Foci Sequester PRC1 and MeCP2 into Cancer-Specific Nuclear Bodies. Cell Rep. 2017 Mar 21;18(12):2943-2956. doi: 10.1016/j.celrep.2017.02.072. PMID: 28329686; PMCID: PMC5429029.
Rabeler C, Paterna N, Potluri R, D'Alessandro LR, Bhatia A, Chen SY, Lee J, Abeje B, Lipchin B, Carone BR, Carone DM. Locus-specific differential expression of human satellite sequences in the nuclei of cancer cells and heat-shocked cells. Nucleus. 2024 Dec;15(1):2431239. doi: 10.1080/19491034.2024.2431239. Epub 2024 Dec 2. Erratum in: Nucleus. 2025 Dec;16(1):2443274. doi: 10.1080/19491034.2024.2443274. PMID: 39620275; PMCID: PMC11622622.
Keywords: Non-coding RNA, Cancer