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Title:Genomic and microscopic dissection of nuclear compartmentalization and function
Author(s):Zhang, Liguo
Director of Research:Belmont, Andrew
Doctoral Committee Chair(s):Belmont, Andrew
Doctoral Committee Member(s):Prasanth, Kannanganattu; Brieher, William; Stubbs, Lisa; Zhao, Huimin
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Genome organization
Nuclear position
Nuclear compartment
Nuclear body
Nuclear speckles
Nuclear lamina
Next generation sequencing
Gene expression
Cell synchronization
Histone modification
Chromatin structure
Chromatin decondensation
Heat shock
Stem cell
Abstract:The spatial genome organization in the cell nucleus is non-random and important for gene expression regulation. Abnormal nuclear organization is related to disease. Microscopy measurements of individual genes reveal a population-based, statistical gradient of increased gene activity from the nuclear periphery to the center. Sequencing-based methods (Hi-C, DamID) suggest the genome could be partitioned into two compartments: transcriptionally active and inactive chromatin positioned at the interior and periphery of the nucleus, respectively. Newer genomic methods (SPRITE, GAM) suggest multi-way interactions occurring more frequently in the nuclear interior, specifically at nucleoli and nuclear speckles. But it is still elusive how such nuclear compartments (bodies) are involved in genome organization. Nuclear speckles are a type of nuclear body enriched in RNA metabolic factors and correlated with active gene expression. A first genome-wide mapping of chromosomal distances from nuclear speckles by a novel genomic method developed in our lab, TSA-Seq, reveals ~5% of the genome deterministically positions at the periphery of speckles, as one major gene expression “hot-zone” in the nuclear interior. Yet the function and dynamics of genome organization relative to nuclear speckles are still unclear. In this dissertation, I applied TSA-Seq combined with microscopy to dissect genome organization relative to nuclear compartments (speckles, lamina, and nucleolus), and its dynamics and function. In Chapter 2, I first improved TSA-Seq with 10-20-fold higher sensitivity (TSA-Seq 2.0) to enable efficient mapping of genome organization relative to nuclear speckles. I applied TSA-Seq 2.0 to mapping nuclear speckles in four human cell lines and in one cell line upon heat shock. We found a largely conserved genome organization relative to speckles with small position changes tightly correlated with gene expression changes. In Chapter 3, I addressed the question of whether transcription is required for speckle association of certain chromatin domains. Speckle TSA-Seq mapping upon transcription inhibition revealed largely unchanged genome organization relative to speckles in either cycling or synchronized early-G1 phase cells, suggesting speckle association is independent of strong transcription. In Chapter 4, I optimized and extended TSA-Seq mapping to nuclear lamina in the same four human cell lines. Regions changing position relative to speckles show opposite changes relative to lamina. But there are many more changes relative to lamina including differential localization of heterochromatin regions between nuclear lamina and nucleolus. Our results reveal an initial 3D picture of nuclear compartmentalization. Further extension of TSA-Seq mapping into multiple nuclear compartments in different cell types and states will help further dissect nuclear organization and function.
Issue Date:2020-03-23
Rights Information:© 2020 Liguo Zhang
Date Available in IDEALS:2020-08-27
Date Deposited:2020-05

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