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Differential regulation of chromosome territory organization in osteoblasts and osteosarcoma

Bisht, Madhoolika
http://rave.ohiolink.edu/etdc/view?acc_num=osu1713508488860584

Abstract Details

2024, Doctor of Philosophy, Ohio State University, Molecular Genetics.
Chromosome organization has a major impact on cellular functions such as transcription, replication, and genome stability. Perturbations to chromatin arrangement impairs cellular functions which in turn causes cell death and cancer. Although organized, the interphase chromatin is dynamic and undergoes rearrangement to allow the much-required flexibility to access DNA for maintaining metabolic and cellular processes. In recent years, several discoveries regarding the higher order conformation have produced contradictory results. These results challenge the previous knowledge regarding the formation of higher order structures and the mechanisms regulating the 3D genome organization. Here, we study the large-scale chromosome territory conformation and organization in the context of diseases and its associated function. We used a CRISPR based live cell imaging technology to fluorescently label chromosome 19 (C19q) and track real-time dynamics of chromosome territory conformation in osteoblast and osteosarcoma cells. We have observed striking differences in chromosome compaction between osteosarcoma and osteoblast. Most osteoblasts have a predominantly extended C19q conformation, whereas osteosarcoma have a more collapsed conformation. The deformation of C19q in osteosarcoma cells is regulated by histone modifications and two major chromatin architectural proteins – CCCTCF-binding factor (CTCF) and cohesin complex. The collapsed C19q conformation is a result of chromatin loops stabilized by CTCF and Cohesin. Upon knocking down CTCF and RAD21 (an essential subunit of cohesin complex) in osteosarcoma, the collapsed C19q undergoes extension at physiological conditions. The extension of C19q conformation in RAD21 knockdown osteosarcoma cells was not reproducible when imaging at non-physiological conditions. This suggests that physiological conditions play a critical role in maintaining correct chromatin conformation and organization. The extended C19q conformation in osteoblasts is a result of structural rigidity provided by the histone post translational modification - H3K27me3. Upon reducing the H3K27me3 by allosteric inhibition of the H3K27me3 writer, we observe that the extended C19q conformation becomes collapsed. This indicates that the different C19q conformation observed in osteoblasts and osteosarcoma are regulated by different mechanisms. We intended to elucidate the functional role of these chromosome conformation differences. To do so, we integrated RNA-seq and ChIP-seq and observed a moderate correlation between H3K27me3, H3K27ac and gene expression. This indicates that although H3K27me3 play a role in maintaining gene expression patterns in osteoblasts, there may be other levels of chromatin architecture involved. Moreover, we did not find a direct relationship between 3D chromosome architecture and transcription as transcription inhibition did not have a noticeable impact on C19q conformation. This indicates a non-linear and a rather complex relationship between 3D genome architecture and gene regulation. Our research employing interdisciplinary techniques provides a unique insight into the complex regulatory mechanisms of large-scale chromatin compaction in normal and disease cells and their impact on gene expression patterns.
Li-Chun Tu (Advisor)
Jian-Qiu Wu (Committee Member)
Robin Wharton (Committee Member)
Mark Parthun (Committee Member)
180 p.
Genetics
Chromosome conformation; CTCF and cohesin; H3K27me3; Osteosarcoma; live-cell imaging

Recommended Citations

Citations

  • Bisht, M. (2024). Differential regulation of chromosome territory organization in osteoblasts and osteosarcoma [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1713508488860584

    APA Style (7th edition)

  • Bisht, Madhoolika. Differential regulation of chromosome territory organization in osteoblasts and osteosarcoma. 2024. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1713508488860584.

    MLA Style (8th edition)

  • Bisht, Madhoolika. "Differential regulation of chromosome territory organization in osteoblasts and osteosarcoma." Doctoral dissertation, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1713508488860584

    Chicago Manual of Style (17th edition)

osu1713508488860584
88
© 2024, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.