Poster abstracts
Poster number 5 submitted by Madhoolika Bisht
Differential regulation of large-scale human chromosome conformations in living cancerous and non-cancerous cells
Madhoolika Bisht (Department of Molecular Genetics, The Ohio State University), Sydney Willey, Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology), Meng Wang, Benjamin Sunkel, Bejamin Stanton (Nationwide Childrens Hospital, Center for Childhood Cancer and Blood Diseases, Columbus, OH 43205, USA ), Li-Chun Tu (Department of Biological Chemistry and Pharmacology, The Ohio State University College of Medicine)
Abstract:
Chromosome conformation has a major impact on gene expression, perturbations to which can impair important cellular functions which in turn lead to cancer. Chromosome compaction is hierarchical. The DNA wraps around core histone proteins to form the chromatin. Chromatin is further compacted by looping to form compartmental domains which eventually leads to formation of chromosome territories that occupy distinct 3D spaces within the interphase nucleus. However, the regulation of chromosome territory conformation in diseases remains understudied. Using CRISPR-SIRIUS, we fluorescently labelled chromosome 19 and tracked real-time dynamics of chromosome territory conformation in live cells. We observed that 86% of osteoblast cells have an extended chromosome 19 territory conformation, whereas in osteosarcoma only 31% of cells demonstrate extended conformation. The collapsed chromosome conformation is a result of increased chromatin loops stabilized by the chromatin architectural proteins CCCTC- binding factor (CTCF) and cohesin complex. Knocking down CTCF and RAD21 (an essential subunit of cohesin complex) leads to the extension of chromosome 19 in osteosarcoma under physiological conditions. We observed that the heterochromatin mark H3K27me3 was largely removed in osteosarcoma cells. Misregulation of H3K27me3 has been reported to repress gene expression and promote collapsed chromosome conformations. Surprisingly, the H3K27me3 mark in normal osteoblasts was essential to maintain extended chromosome conformation as the extended chromosome conformation collapsed in normal osteoblasts upon removal of H3K27me3 marks. Integration of RNA-seq and ChIP-seq data reveals the role of H3K27me3 in regulating gene expression in osteosarcoma. ~90% of upregulated genes in osteosarcoma also demonstrated loss of H3K27me3. In summary, our study not only shows the distinction between chr 19 conformation but also shows that we can regulate the chromosome territories conformation in osteosarcoma and osteoblasts. Our research employs interdisciplinary techniques that provide a unique insight into the complex regulatory mechanisms of large-scale chromatin compaction in cancer cells.
Keywords:
Keywords: Chromosome conformation, H3K27me3, CTCF, Cohesin, Live-cell imaging