New insights into 3D genome architecture through improved genomic methods

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Cell and Developmental Biology

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Kumar, Pradeep

Contributors dc:contributor

• Belmont, Andrew S.
• Prasanth, Kannanganattu V.
• Freeman, Brian C.
• Kalsotra, Auinash

• 3D genome organization
• Nuclear compartment
• Nuclear speckles
• Nuclear lamina
• Nucleolus

Statement dc:rights

• Copyright 2025 Pradeep Kumar

Language dc:language

en, eng

Handle dc:identifier

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

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129695