Functional Interactions Between Chromatin Modifying Complexes and the Nuclear Pore in the Yeast Saccharomyces cerevisiae Open Access

Kerr, Shana (2010)

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The nucleus is functionally organized by the arrangement of the chromosomes,
the nuclear periphery, and spatial regulation of transcription. Though the nuclear
periphery has been historically viewed as transcriptionally repressive, recent work in the
budding yeast Saccharomyces cerevisiae has revealed that some genes physically relocate
from the nuclear interior to the nuclear periphery upon transcriptional activation, where
they associate with nuclear pore complexes (NPCs). This dissertation focuses on
elucidating the mechanism and physiological significance of this phenomenon.
Our work reveals functional interactions between actively transcribed genes,
chromatin modifying complexes, and the NPC. Specifically, we identify the SAGA
histone acetyltransferase as a necessary link between the NPC and active GAL genes.
Interestingly, this association requires the physical presence of SAGA rather than
transcriptional activation by SAGA, suggesting that gene interaction with the NPC is
mediated by protein-protein interactions between NPC subunits and transcriptional
activators. Our studies also reveal that functional interactions between SAGA and the
NPC regulate global transcript levels, particularly for highly transcribed genes. These
findings suggest a role for NPC-gene interactions in regulating the global transcription of
highly induced genes. In addition, we find that interactions between NPC and SAGA
subunits are required for the retention of the GAL1 gene at the NPC, and defects in gene
retention due to loss of NPC and SAGA subunits correlate with reduced ability of these
cells to metabolize galactose. These findings suggest that gene relocation is comprised of
two steps, recruitment and retention, and that gene relocation makes a significant
contribution to transcriptional regulation. We also identify new factors potentially
involved in gene relocation based on functional interactions between INO80 chromatin
remodeling complex components and NPC subunits. Interestingly, we find that
interactions between the NPC and INO80, as well as interactions between the NPC and
SAGA, may play a role in DNA damage repair. These observations are consistent with a
physiological role for relocation of damaged DNA to the NPC, analogous to relocation of
transcribed genes. Taken together, these results suggest that the NPC is an important
regulator of chromatin dynamics that promotes an open chromatin structure permissible
to active DNA transactions.

Table of Contents

Table of Contents

Chapter 1

Introduction and Background 1
Overview of Nuclear Organization 2
The Nuclear Pore Complex 5
DNA Transactions at the NPC 9
Characterized Loci and Mechanisms of Gene Relocation to the NPC 16
Physiological Relevance of Gene Interactions with the NPC 23
Scope of this Dissertation 26

Chapter 2

Actively Transcribed GAL Genes can be Physically Linked to the Nuclear Pore by the SAGA Chromatin Modifying Complex 30
Introduction 31
Results 34
Discussion 48
Experimental Procedures 50

Chapter 3

The SAGA Complex and the Nuclear Pore Control Global Transcription and Mediate Retention of the GAL1 Locus at the Nuclear Periphery 56
Introduction 57
Results 61
Discussion 85
Experimental Procedures 88

Chapter 4

Functional Interactions between Chromatin Modifying Complexes and the Nuclear Pore Play a Role in DNA Damage Repair 94
Introduction 95
Results 98
Discussion 108
Experimental Procedures 110

Chapter 5

Conclusions and Discussion 113
The Mechanism of Gene Relocation to the NPC 114
The Physiological Relevance of Gene Relocation to the NPC 119
Evolutionary Conservation of Nup Association with Active Genes 121
Final Conclusions 125

References 126

The Ccr4-Not Complex Physically and Functionally Interacts with the mRNA Export Pathway 155
Introduction 156
Results 159
Discussion 172
Experimental Procedures 177
References 181

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