Nuclear speckle dynamics and function of speckle association in gene expression

Nuclear speckles have been studied intensively for a long time. Accumulating evidence has suggested that nuclear speckles have a dynamic structure, and are associated with a significant fraction of active chromosome regions and genes. However, their mobility and the regulation mechanism of their number and size are still poorly understood. Importantly, the functional significance of the speckle-gene association also remains unclear. To understand speckle dynamics and effects of speckle-gene association on gene expression better, I study the mobility of nuclear speckle bodies and its effect on morphological change of speckle size in chapter 2, and transcription enhancement by speckle-gene association in chapter 3. In chapter 2, I show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2-1.5 µm/min. Frequently, 3 or even 4 speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to the formation of fewer but larger speckles after transcriptional inhibition. These speckle movements are not actin-dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle-marker protein SON. Our observations suggest a mechanism for long-range, directed nuclear speckle movements, contributing to the overall regulation of nuclear speckle number and size as well as the nuclear organization. In chapter 3, I show nuclear speckle association results in several-fold transcriptional amplification of Hsp70 genes. Hsp70 BAC transgenes and endogenous genes turn on 2-4 mins after heat shock irrespective of their distance to nuclear speckles. However, I observe 12-56-fold and 3-7-fold higher transcription levels for speckle-associated Hsp70 transgenes and endogenous genes, respectively, after 1-2 hrs heat shock. Several-fold higher transcription levels for several genes flanking the Hsp70 locus also correlate with speckle-association at 37 ℃. Live-cell imaging reveals this modulation of Hsp70 transcription temporally correlates with speckle association/disassociation. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment enriched in RNA processing and transcription-related factors through a “transcriptional amplification” mechanism, which is distinct from transcriptional bursting.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2019

Author dc:creator

• Kim, Jiah

Contributors dc:contributor

• Belmont, Andrew S.
• Zhao, Huimin
• Prasanth, Kannanganattu V.
• Brieher, William M.

• Nuclear speckle, liquid droplet, smRNA FISH, transcription, live-cell imaging

Statement dc:rights

• Copyright 2018 Jiah Kim

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/102813

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/102813