New insights into 3D genome architecture through improved genomic methods

Kumar, Pradeep

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https://hdl.handle.net/2142/129695 Copy

Description

Title

New insights into 3D genome architecture through improved genomic methods

Author(s)

Kumar, Pradeep

Issue Date

2025-04-28

Director of Research (if dissertation) or Advisor (if thesis)

Belmont, Andrew S.

Doctoral Committee Chair(s)

Belmont, Andrew S.

Committee Member(s)

• Prasanth, Kannanganattu V.
• Freeman, Brian C.
• Kalsotra, Auinash

Department of Study

Cell & Developmental Biology

Discipline

Cell and Developmental Biology

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

3D genome organization Nuclear compartment Nuclear speckles Nuclear lamina Nucleolus

Abstract

The eukaryotic interphase nucleus contains morphologically and functionally distinct substructures known as chromatin domains and nuclear bodies. Genome organization within the nucleus is highly nonrandom, and abnormal changes in genome organization can lead to diseases including cancer. Two widely used models in the field: the radial genome organization model and the binary division model address various aspects of genome organization. However, while these models are effective in explaining certain features, they have significant limitations, including their failure to account for the nuclear bodies and their possible influence on genome organization. Lamina-associated domains (LADs) localize not only at nuclear lamina but also localize at the nucleolus and pericentromeric heterochromatin (PCH). In Chapter 2, I present nucleolus and centromere TSA-seq methods, which were developed to map genomic regions localizing at the nucleolus and PCH. Using these methods, I made several interesting observations about genomic regions localizing at these nuclear substructures, including the identification of two distinct subsets of LADs that exhibit unique gene expression profiles and DNA replication timing, despite their differential localization at the nucleolus or nuclear lamina, depending on the cell type. Unlike previous studies in the field, Chapter 3 presents my collaborative research where we integrated multiple spatial (DamID, TSA-seq and Hi-C), functional genomic methods (16-fraction Repli-seq and RNA-seq), combined with light microscopy, to investigate genome organization in relation to various nuclear bodies across multiple cell types. This work uncovered several novel features of genome organization and challenges long-standing models in the field. Finally, the appendices of this thesis, I include several protocols for novel genomic methods. These include a new protocol for the TSA-seq nanotag (TSA-seq 3.0) method, which requires 500 times fewer cells compared to the current TSA-seq 2.0 protocol and has higher throughput. Additionally I present an improved nanotag protocol that addresses the limitations of the CUT&Tag method and is compatible with fixed cells.

Graduation Semester

2025-05

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/129695

Copyright and License Information

Copyright 2025 Pradeep Kumar

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