Investigation of RNA-mediated Chromatin Organization in Normal and Disease State Restricted; Files Only

Abstract

Satellite RNAs, typically repressed in normal cells, exhibit significant upregulation in certain cancer types, suggesting their involvement in tumorigenesis. To understand their expression pattern in normal and various cancers, we analyzed RNA sequencing (RNA-seq) from normal mouse and human tissues and compared them with tumor datasets from glioblastoma, pancreatic, breast, and lung cancers. Our analysis revealed minimal satellite RNA expression in normal tissues, whereas tumors displayed highly heterogeneous and cancer-type-specific upregulation. Glioblastoma exhibited the highest levels of satellite RNA, aligning with its high degree of genomic instability. To investigate the functional impact of satellite RNA dysregulation, we first assessed its role in chromatin organization in normal cells. CUT&RUN with in situ RNase treatment showed that RNA depletion significantly reduced CUT&RUN DNA recovery for H3K27me3, indicating that RNA plays an architectural role in maintaining H3K27me3 heterochromatin. We found that the reduced recovery of H3K27me3 heterochromatin CUT&RUN signal resulted from reduced antibody and Micrococcal nuclease access to H3K27me3 heterochromatin. These results suggest for the first time that RNA impacts chromatin organization and, thereby, the access to various chromatin interacting factors. Our research findings establish consistent satellite RNA dysregulation across multiple cancer types in both humans and mice, suggesting its potential role in chromatin remodeling and tumorigenesis. Further studies would focus on how RNA dysregulation, including satellite RNA, impacts the access of chromatin-associated factors in various cancer types and explore its potential as a biomarker for early cancer detection.

Table of Contents

Introduction ................................................................................................................. 1

Figure 1. Centromeric and Pericentric Satellite Map in Human and Mouse .............................. 3

Materials and Methods .................................................................................................. 6

Results ....................................................................................................................... 11

Figure 2. Satellite RNA Transcript Levels in Centromeric and Pericentric Regions .................. 12

Figure 3. Comparative Analysis of Satellite RNA Expression in Human Tumor and Normal

Tissues ............................................................................................................................. 14

Figure 4. Differential mRNA Expression of Centromere- and Pericentromere-Associated Proteins

Across Cancer Types. ......................................................................................................... 16

Figure 5. Experimental Workflow for CUT&RUN with In Situ RNase Treatment and Efficiency

Assay ............................................................................................................................... 18

Figure 6. CUT&RUN Enrichment Profiles and Library Concentration Changes ..................... 19

Figure 7. Western Blot Analysis of Antibody Binding Across RNA Concentrations ................... 21

Figure 8. MNase Digestion Efficiency Across RNase Concentration Groups ............................ 23

Figure 9. RNA Depletion Leads to Localized Changes in Chromatin Accessibility at Promoter

Regions ............................................................................................................................ 24

Discussion .................................................................................................................. 24

References ................................................................................................................. 27

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Biology, Genetics Biology, General
Keyword	Chromatin Cancer Non-coding RNA
Committee Chair / Thesis Advisor	Jitendra Thakur, Emory University
Committee Members	Jang Young, Emory University Nic Vega, Emory University

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