Investigation Of The Behavior Of The Gal4 Inhibitor Gal80 Of The GAL Genetic Switch In The Yeast

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Sudip Goswami, M.S.

Graduate Program in

The Ohio State University 2014

Dissertation Committee

Dr. James Hopper, Advisor Dr. Stephen Osmani Dr. Hay-Oak Park Dr. Jian-Qiu Wu

ii

Copyright by

Sudip Goswami

2014

iii ABSTRACT The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a -responsive switch for the GAL of Saccharomyces cerevisiae. Gal4 binds to upstream activation sequences or UASGAL sites on GAL promoters as a dimer both in the absence and presence of galactose. In the absence of galactose, a Gal80 dimer binds to and masks the Gal4 activation domain, inhibiting its activity. In the presence of galactose, Gal3 interacts with Gal80 and relieves Gal80’s inhibition of Gal4 activity allowing rapid induction of expression of GAL genes. In the first part of this work (Chapter 2) in-vitro chemical crosslinking coupled with SDS PAGE and native PAGE analysis were employed to show that the presence of Gal3 that can interact with Gal80 impairs Gal80 self association. In addition, live cell spinning disk confocal imaging showed that dissipation of newly discovered Gal80-2mYFP/2GFP clusters in galactose is dependent on Gal3’s ability to interact with Gal80. In the second part (Chapter 3), extensive analysis of Gal80 clusters was carried out which showed that these clusters associate strongly with the GAL1-10-7 locus and this association is dependent on the presence of the UASGAL sites at this locus. Moreover, the formation of Gal80 clusters is dependent on the presence of Gal4. Additionally, it was discovered that Gal4-2GFP/2mYFP makes intranuclear foci, which unlike Gal80-2mYFP/2GFP clusters do not dissipate in galactose and their formation is not dependent on the presence of Gal80. Similar to Gal80 clusters, these Gal4 foci associate strongly with the GAL1-10-7 locus. These evidences suggest that large molecular assemblies of Gal80 and Gal4 associate with the UASGAL sites of the GAL1-10-7 locus, which raises some interesting possibilities as to how this genetic switch is regulated.

ii

I dedicate this work to the “Future”

iii

Acknowledgements:

I would like to thank Prof. James Hopper, my thesis advisor, for his continuous guidance and advice. I would also like to thank my advisory committee members

Prof. Stephen Osmani, Prof. Hay-Oak Park and Prof. Jian-Qiu Wu for their critical and important input into my work. I thank all my present J. Hopper lab members, Eric

Jiang, Alexander DiScenna and Pallavi Chandna for their technical assistance. I also acknowledge the technical assistance I received from former lab members Kathleen

Dotts, Emily Baas, Jill Steinbrunner, Caitlin Rigsby, and others. I would also like to thank former graduate student Dr. Onur Egriboz with whom I collaborated quite a bit and also for his ideas and input to my work, former post docs Dr. Xiarong Tao and Dr. Fenglei Jiang for their help, and former lab mate Jin Shuo with whom I did my first project in the lab. I also acknowledge many students and post docs of other labs for their generous help and cooperation. I am also grateful to Eric Jiang of the

Jim Hopper lab and Joey Marquadt of the Harold Fisk lab for their critical reading of this thesis.

iv

Vita

1995 ……………………………………………………...B.S. Pharmaceutical Technology Jadavpur University Kolkata, West Bengal, India

2007………………………………………………………M.S. Biotechnology West Bengal University of Technology Kolkata, West Bengal, India

2007-2014……………………………………………..Graduate Associate, Department of Molecular Genetics The Ohio State University

Publication

Egriboz O, Goswami S, Tao X, Dotts K, Schaeffer C, Pilauri V, Hopper JE. 2013 Self- association of the Gal4 inhibitor Gal80 is impaired by Gal3: evidence for a new mechanism in the GAL gene switch Mol Cell Biol. Sep;33(18):3667-7

Field of Study

Major Field: Molecular Genetics

v

Table of contents

Abstract ...... ii

Dedication ...... iii

Acknowledgement ...... iv

Vita ...... v

Table of Contents ...... vi

List of Figures ...... xii

List of Tables ...... xv

List of Abbreviations ...... xvii

Chapter 1 Regulation of and The GAL genetic switch ...... 1

1.Introduction ...... 2

1.2. Main Factors In Transcription Regulation ...... 3

1.2.1Core elements ...... 4

1.2.2 Basal Transcription factors ...... 4

vi

1.2.3Enhancers ...... 4

1.2.3.1 Action at a distance-DNA looping ...... 5

1.2.4 Activators ...... 11

1.3 Genetic regulatory network of yeast galactose pathway ...... 15

1.3.1 for galactose utilization ...... 16

1.3.2 Gal4 – the transcriptional activator ...... 17

1.3.3 Gal80 – the transcriptional inhibitor ...... 21

1.3.4 Gal3 – the galactose sensor and signal transducer ...... 23

1.3.5 The current model of GAL gene switch ...... 23

2. Chapter 2: Interaction of Gal3 with Gal80 impairs

Gal80 self-association

...... 27

2.1 Abstract ...... 28

2.2 Introduction ...... 29

2.3 Materials and Methods ...... 31

2.3.1 Yeast strains and plasmids ...... 31

2.3.2 Microscopy ...... 32

2.3.3 Protein expression and purification ...... 33

2.3.4 Cross-linking of Gal80 and Gal3 ...... 34

vii 2.3.5 Discontinuous blue native protein gel electrophoresis ...... 34

2.4 Results ...... 35

2.4.1 Gal80-Gal80 self-association is impaired under conditions supporting Gal3-Gal80 interaction ...... 35

2.4.2 The nuclei of live cells display clusters of Gal80-2mYFP that dissipate in response to galactose-triggered Gal3-Gal80 interaction

...... 37

2.4.3 Super Gal80S-2 forms more stable higher order oligomers on native gel compared to WT Gal80

...... 38

2.5 Discussion ...... 39

3. Chapter 3 Investigation Of The Intranuclear Clusters Of Gal80 And Intranuclear Foci of Gal4 ...... 51

3.1 Abstract ...... 52

3.2 Introduction ...... 53

3.3 Materials and Methods ...... 54

3.3.1 Yeast strains, plasmids, media and growth condition ...... 54

3.3.2 Immunoblotting ...... 56

3.3.3 Microscopy ...... 57

viii 3.3.4 Counting the number of Gal80-2GFP clusters or

Gal4-2GFP/2mYFP foci per cell ...... 58

3.3.5 Standard for true colocalization ...... 59

3.3.6 Estimation of the number of molecules in the Gal80 clusters and

in the Gal4 foci using fluorescent intensity measurements

...... 60

3.3.7 Measuring distances between the two fluorescent dots of

Tet repressor GFP (TetR-GFP) and LacI-mCherry

...... 62

3.4 Results ...... 63

3.4.1Gal80-2GFP predominantly makes 1, 2, or 3intranuclear clusters

per cell

...... 63

3.4.2 Gal80-2GFP clusters are observed in raffinose but not in

...... 64

3.4.3 Preexisting Gal80 can reassemble into cluster upon

galactose depletion

...... 65

3.4.4 Gal80 clusters colocalize with the UASGAL1-10-7 locus ...... 67

ix 3.4.5 Deletion of UASGAL sites in GAL1-10-7 does not affect the number

distribution of Gal80-2GFP cluster within the yeast population

...... 70

3.4.6 Gal80-2mYFP cluster formation is dependent on Gal4 ...... 70

3.4.7 What is the nature of requirement of Gal4 for

Gal80 cluster formation?

...... 71

3.4.8 PGAL4 DBD-Gal80 can make intranuclear cluster in the absence of both Gal4 and Gal80

...... 72

3.4.9 Is DNA binding required for formation of PGal4 DBD-Gal80-2mYFP clusters?

...... 72

3.4.10 Does occurrence of Gal80-2GFP clusters

depend on Gal80 self-association?

...... 74

3.4.11 Gal4-2GFP and Gal4-2mYFP make intranuclear foci both in the absence and presence of galactose

...... 74

x 3.4.12 Formation of Gal4-2GFP/2mYFP foci is not dependent on Gal80

...... 75

3.4.13 Gal4 makes intranuclear foci independent of Gal80 but are they of equal intensities in the presence and absence of Gal80?

...... 76

3.4.14 The brightest Gal4-2GFP focus colocalizes

with the GAL1-10-7 locus

...... 76

3.4.15 Gal80 clusters and Gal4 foci can be simultaneously observed in the same cell

...... 77

3.4.16 Do the large molecular assemblies of Gal80 constitute bridges between distant UASGAL sites?

...... 78

3.4.17 Is Gal80 clustering correlated with tighter repression? ...... 81

3.5 Discussion ...... 85

References ...... 149

xi

List of Figures

1.1 Functional domains of Gal4 protein ...... 25

1.2 Model of the GAL gene switch at the time of initiation of this thesis work ...... 26

2.1 Gal3’s capacity to impair Gal80 self-association depends on its capacity to bind to Gal80 ...... 42

2.2 Increasing levels of Gal3 binding reduced cross-linked Gal80 molecules in an ATP/Galactose dependent manner ...... 44

2.3 Gal3’s capacity to bind to Gal80 is required for galactose-triggered dissipation of nuclear Gal80 clusters ...... 46

2.4 The Gal80S-2 variant that is defective in binding to Gal3

shows more prominent multimerization than the wild type Gal80

...... 50

3.1 Gal80-2GFP exhibits predominantly 1, 2, or 3 intranuclear clusters

...... 100

3.2 Gal80-2GFP clustering is observed in raffinose but not in glucose ... 102

xii 3.3 Preexisting Gal80-2GFP can reassemble into cluster upon galactose depletion

...... 104

3.4 Gal80 clusters colocalize with the GAL1-10-7 locus ...... 110

3.5 Gal80 clusters colocalize with the GAL1-10-7 locus ...... 114

3.6 Deletion of UASGAL sites in GAL1-10-7 does not affect the number distribution of Gal80-2GFP cluster in yeast population ...... 117

3.7 Gal80-2mYFP cluster formation is dependent on Gal4 ...... 119

3.8 Requirement of Gal4 for Gal80-2mYFP cluster formation can be bypassed by providing Gal80 with the Gal4DBD ...... 121 3.9 PGal4 DBD-Gal80-2mYFP can make intranuclear cluster in the absence of both Gal4 and Gal80 but SV40 NLS–Gal80-2mYFP cannot ...... 123 3.10 Gal80 mutant Gal80N230R defective in dimerization does not make cluster ...... 125 3.11 Gal4-2GFP appears as intranuclear foci both in the absence and presence of galactose ...... 127

3.12 Gal4 foci formation is not dependent on Gal80 ...... 129

3.13 The brightest Gal4-2GFP focus colocalizes with the GAL1-10-7 locus ...... 131

3.14 Gal80 cluster and Gal4 foci can be simultaneously observed in the same cell ...... 133

xiii

3.15 Schematic depiction of the general scheme/approach to visualize two loci simultaneously as two fluorescent dots of different fluorescent in the cell ...... 135

3.16 Scheme showing the general strategy to observe real-time synthesis of transcript at a given locus ...... 136

3.17 Simultaneous monitoring of Gal80-2mYFP clusters and the transcription status of a given locus ...... 137

xiv

List of Tables

2.1 Plasmids used in this study ...... 32

3.1 Distribution of percentages of cells with a specific number of Gal4-2GFP/2mYFP foci per nucleus ...... 139

3.2 Analysis of colocalization between Gal4-2GFP foci and Gal80-tdTomato cluster in the WT GAL1-10-7 strain ...... 140

3.3 Analysis of colocalization between Gal4-2GFP foci and Gal80-tdTomato cluster in the ΔGAL1-10-7 strain ...... 140

3.4 There is no significant difference between the distances between two GAL1 loci and one GAL1 locus with a LEU2 locus (without nocodazole treatment) ...... 141

3.5 There is no significant difference between the distances between two GAL1 loci and one GAL1 locus with LEU2 locus (p values of KS test) ...... 141

3.6 There is no significant difference between the distances between two GAL1 loci and one GAL1 locus with LEU2 locus (with nocodazole) ...... 141

3.7 There is no significant difference between the distances between two GAL1 loci and one GAL1 locus with LEU2 locus (p values KS test) ...... 141

3.8 Distribution of cells with respect to cluster colocalization to the LacOx64 array and appearance of transcript dots ...... 142

xv 3.9 Yeast strains used in this study ...... 143

3.10 Plasmids used in this study ...... 147

xvi

List of Abbreviations

E. coli, BRE, TFIIB Recognition Element MTE, Motif Ten Element DPE, Downstream Promoter Element DCE , Downstream Core Element XCPE1, X Core Promoter Element TBP, TATA-box binding protein Shh, Sonic hedgehog LCR, locus control region Ifng, interferon γ PIC, pre-initiation complex ORF, open reading frame DBD, DNA binding domain AD, Activation domain UAS, Upstream activation sequence aa , amino acid S. cerevisiae, Saccharomyces cerevisiae K lactis, Kluyveromyces lactis WT, wild type DVR, Divergent region (GAL1-10) NLS, Nuclear localization signals SV40, Simian virus 40 GFP, Green fluorescent protein YFP, Yellow fluorescent protein CFP, Cyan fluorescent protein mYFP, monomeric YFP TR, Tet Repressor SD, Standard deviation SEM, Standard error of mean KS test , Kolmogorov-Smirnov Test FISH, fluorescence in-situ hybridization

xvii

Chapter 1:

Regulation of Transcription and The GAL genetic switch

1

1.1 INTRODUCTION:

Gene expression is the process by which information encoded in a gene is used to synthesize a functional gene product. Genes may carry information for various types of end products. A protein-coding gene carries the information for synthesis of protein whereas the non-protein coding genes contain information for RNA final products such as ribosomal RNA (rRNA), transfer RNA (tRNA), long noncoding RNA

(ncRNA), microRNA, small interfering RNA etc. Complex interplay between these gene products and the environment (both internal and external) determines the adaptability and survivability of the organism. It is therefore of paramount importance that be tightly regulated. Not surprisingly, living organisms have evolved exquisite and elaborate control systems to regulate their gene expression in critical, adaptive ways. This introduction will focus on those aspects of regulation of protein-coding genes that relate to this study and a brief introduction to the GAL genetic switch in the budding yeast Saccharomyces cerevisiae.

In the studies presented in this work, the GAL genetic switch was used as a model system to gain insights into the transcriptional regulation in Saccharomyces cerevisiae. More specifically, in this work, two central questions have been addressed. The first question relates to the role of the Gal3-Gal80 interaction on 2 Gal80 self-assemblies. This work is covered in chapter 2. Chapter 3 will address the second question, which is related to the basis of the formation of the newly discovered intranuclear clusters of Gal80-2GFP/2mYFP (introduced in chapter 2) and possible functions of these clusters. A common aspect to both of these questions is the role of self-assemblies of proteins in transcriptional regulation and to some degree how protein-protein interactions may facilitate intra and/or interchromosomal associations and the role of such associations in regulation of transcription. In this review, I will first present the literature related to both of these aspects after a brief introduction to the key players in .

Secondly, since I have utilized the GAL genetic switch as a model system for my studies, I will briefly discuss about the key regulatory players of this switch in the second part.

1.2 MAIN FACTORS IN TRANSCRIPTION REGULATION: Transcriptional regulation of genes involves proteins that facilitate recruitment of RNA to the relevant gene promoters in response to environmental and/or developmental signals. In both and , the main factors for transcriptional regulation can be broadly grouped into two classes: cis-acting DNA elements and trans-acting protein factors. In eukaryotes, cis-acting DNA elements can be further subdivided into core promoter elements, enhancers, insulators etc. whereas the trans-acting protein factors could be subdivided into basal transcription factors, activators, coactivators, , etc. This work is primarily related 3 to the roles of enhancers and activators in transcriptional regulation. Therefore, I will focus on the roles of the enhancers and the activators in transcriptional regulation.

1.2.1 Core promoter elements: The core regulatory DNA element for eukaryotic transcription is the core promoter, a DNA region that directs accurate initiation of transcription by RNA PolII. The core promoter contains different sequence motifs such as the TATA box, BRE (TFIIB Recognition Element), Inr (Initiator), MTE (Motif

Ten Element), DPE (Downstream Promoter Element), DCE (Downstream Core

Element), XCPE1 (X Core Promoter Element) etc. (Juven-Gershon and Kadonaga

2010 and references therein) that play important roles in the initial events of transcription initiation.

1.2.2 Basal Transcription factors: Purified RNA Pol II cannot distinguish promoter from a non-promoter DNA. It requires additional factors for transcription initiation termed as general or more appropriately basal transcription factors. These include

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.

1.2.3 Enhancers: Enhancers, the promoter-distal sequence elements, play a key role in the regulation of gene expression in eukaryotes. One key feature of enhancers is the flexibility of their location and orientation relative to the promoter. In S. cerevisiae, UAS’s (upstream activation sequences) are involved in transcription 4 activation through the binding of transcriptional regulatory proteins. These share similarities with sequences of metazoans as they both mediate transcription activation in either orientation and at variable distances from the target gene promoters (Guarente et al. 1984). This similarity makes Gal4, a transcriptional activator that binds to specific DNA sequences termed as UASGAL, an extremely useful tool to study how transcriptional regulators work in conjunction with enhancers or similar cis-acting elements to mediate transcriptional response.

1.2.3.1 Action at a distance – DNA looping: As mentioned above, one key feature of the enhancers is the variability in their location and orientation relative to the promoters of the genes they regulate. They are often found a great distance away from the gene they regulate, for example the limb bud enhancer for the mouse Sonic hedgehog (Shh) gene is located within another gene more than 1Mb (mega base pairs) from the Shh gene promoter (Bulger and Groudine 2011 and references therein). Another example is the locus control region or LCR, a group of sequence elements distributed over a region of 20-30 kb (kilo base pairs) located upstream of a gene cluster and required for high level of expression of mammalian β-globin genes in erythroid cells (Bulger and Groudine 2011 and references therein). One important question is how enhancers communicate with their target promoters.

Although there are several mechanisms proposed, recent studies employing RNA

TRAP (Holwerda and de Laat 2012 and references therein) or genome-wide chromosome conformation capture (3C) and their more sophisticated variants indicate that predominant mechanism is DNA looping where the promoters and the 5 enhancers are brought in close proximity through protein-protein interactions where the intervening DNA sequences are looped out or otherwise organized to permit such interactions (Bulger and Groudine 2011 and references therein).

DNA looping is associated with many cellular processes such as transcription, recombination, and replication (Adhya 1989, Schlief 1992, Matthews 1992, Vilar and Saiz 2005), allowing distant DNA regions to associate. It is particularly prominent in the regulation of gene expression. DNA looping was first identified in the Escherichia coli (E. coli) ara (Dunn et al. 1984) where in the absence of arabinose (a carbon source for E. coli), two molecules of the main regulatory protein

AraC bound to two distant operator sites araO2 and araI1 (separated by 211 base pairs (bp)) self associate through DNA looping to form a dimer that leads to repression of expression of genes involved in utilization of arabinose as a carbon source (Schlief 2002) as well as expression of AraC itself. Similar looping was later discovered in the lac and gal in E. coli (Adhya 1989, Schlief 1992). In the , the (LacR or LacI) binds as a dimer to the primary operator site O1; however, it was proposed that at correct helical phasing, LacR dimers bound to auxiliary operator sites O2 or O3 (O1 and O2 are separated by 401 bp, O1 and O3 are separated by 92 bp) could associate with the LacR dimer bound to the primary operator site O1 to form a tetramer through DNA looping. It has been postulated that this association increases the effective local concentration of the LacR at the primary operator site O1 and confers a stronger repression on the lac gene transcription (Becker 2005). In the , the repressor GalR dimers bound at 6 two distant operator sites OE and OI flanking the gal promoters self-associate to form tetramers via DNA looping and this loop is stabilized by an architectural protein HU. This higher order nucleoprotein complex referred to as gal

“repressosome” (repressosome hypothesis) represses the transcription of gal genes in E. coli (Aki and Adhya 1997, Lewis and Adhya 2002). The newly discovered

Gal80-2GFP/2mYFP clusters seems to represent higher order self-assemblies of

Gal80 and one of the hypotheses tested in this work is whether these clusters are functioning as a repressosome where their association with the GAL genes results in tighter repression as seen in the gal operon in E. coli.

DNA loops formed both in the ara and lac operons are examples of short loops. In the lac operon, it was observed that the influence of the auxiliary operators on the strength of repression decreases with increasing inter-operator distances and was almost nonexistent at a distance of 1000 bp (Revet et al. 1999 and references therein). This raised the question as to how can these loops be formed and maintained over a distance of several kilobases. Revet and colleagues (1999) used bacteriophage λ repressor cI to address this question. Bacteriophage λ infects E. coli and upon infection it chooses either to replicate by lytic growth or to enter a latent state termed as a “lysogenic state or lysogeny” in which phage DNA is integrated into that of the bacterial host. In the state of lysogeny, the only phage gene that is expressed is that which encodes the repressor cI, which plays the central role in the lysis-lysogeny switch. The λ repressor binds to a series of six operator sites organized into two operator regions, OR and OL as a dimer and regulates the 7 activities of several promoters critical for the decision between lytic and lysogenic growth (Koudelka 2000 and references therein). The λ repressor is a dimer in solution at wild-type (WT) intracellular concentration (Revet et al. 1999 and references therein) and the two dimers can form DNA loops between two operators separated by five or six helical turns (Revet et al. 1999 and references therein). Two

λ repressor dimers bind cooperatively to two adjacent operators separated by 24 bp, and these adjacent dimers can form tetramers through interactions between their non-DNA-binding carboxy-terminal domains (Revet et al. 1999 and references therein). Free λ repressor forms octamers only at a very high nonphysiological concentration (Revet et al. 1999 and references therein). Revet et al. (1999) hypothesized that λ repressors can octamerize even at physiological concentrations when DNA is provided as a proper scaffold to form tetramers by cooperative binding of two dimers on adjacent operators. Using electron microscopy (EM) the authors showed that λ repressor tetramers bound to operator sites separated by

2850 bp or 2470 bp could associate to form octamers with the intervening DNA looped out. They further showed that such long-distance protein-protein interaction and formation of higher order structure in the context of λ repressor is important for transcriptional repression. Protein-protein interaction between Gal80 molecules bound to Gal4 on adjacent UASGAL sites had been proposed to be important for tight repression of GAL genes (Melcher and Xu 2001). In this study it was found that the

Gal80 clusters strongly associate with UASGAL sites and they also seem to represent self-assemblies of Gal80 molecules as mentioned previously. Therefore, it would be interesting to know if formation of such higher order structure of Gal80 associated 8 with DNA (Gal80 binds to Gal4 bound to UASGAL) is also playing a critical role in the

GAL gene regulation.

Formation of homo-multimers or hetero-multimers through associations of proteins bound to distant DNA sites has also been observed in humans. For example, using

EM as well as scanning transmission EM (STEM), Stenger et al. (1994) showed that tumor suppressor protein p53 bound to its cognate (RE) could assemble oligomers (homomultimers) by two distinct interactions. Through its C- terminus tetramerization domain, p53 forms tetramers (Stenger et al. 1994 and references therein) and through a separate oligomerization domain these tetrameric p53 bound to distal DNA sites (RE sites) interact to form multimers through the interaction termed by the authors as “stacking interaction” (Stenger et al. 1994). This multimerization has been shown to be important for maximal effect of p53 on its target gene expression possibly by increasing the local concentration of p53 (Stenger et al. 1994). In this study it was observed that Gal4-2GFP/2mYFP expressed from its endogenous locus associates very strongly with the GAL1-10-7 locus, and it also appears that there are more molecules of Gal4 associating with the locus than what could theoretically be expected (discussed in detail in Chapter 3).

Although there has been no direct evidence in the literature for Gal4 oligomerization beyond the dimeric state (Carey et al. 1989, Marmorstein et al.

1992), the presence of what seems to be large assembly of Gal4 molecules raises the possibility that Gal4 might indeed multimerize beyond the well-known dimeric state

(Carey et al. 1989, Marmorstein et al. 1992). Interestingly, Gal4 middle region does 9 contain a predicted coiled-coil domain (ExPasy Bioinformatics Resource Portal; program: COILS; http://embnet.vital-it.ch/software/COILS_form.html) in addition to the well-known strong coiled-coil domain at the N-terminus (amino-terminus)

(Carey et al. 1989, Marmorstein et al. 1992). Given the well-documented roles of the coiled-coil domain in protein oligomerization (Burkhard et al. 2001 and references therein), it will be interesting to know if the predicted coiled-coil domain of Gal4 middle region is mediating Gal4 multimerization and if mechanisms similar to what was observed with p53 may also be playing an important role in GAL gene regulation.

In addition to the long-range intrachromosomal interactions described above, there have been reports of interchromosomal associations playing a regulatory role in gene expression. One system where such interchromosomal association has been observed is in the differentiation of naïve CD4+ T helper cells into TH1 and TH2 cells. In mice interferon γ (Ifng), the cytokine gene for TH1 is located on chromosome 10, whereas the cytokine genes for TH2 cells (interleukin 4, 5 and 13) and their LCR are located in a gene cluster on chromosome 11 (Williams et al. 2010 and references therein). In naïve CD4+ T cells where there is no expression of any of these cytokines, interchromosomal association between the regulatory regions of the TH2 cytokine locus and Ifng promoter locus was observed. Differentiation to either TH1 or TH2 results in expression of specific cytokine along with the loss of the interchromosomal association between the LCR of TH2 specific cytokine gene 10 cluster (interleukin 4, 5, and 13) and the promoter region of the Ifng gene (Williams et al. 2010 and references therein). It has been proposed that such associations poise the two classes of cytokine genes for rapid expression upon T cell receptor stimulation. It was observed that mutations within the TH2 LCR not only affect the expression of TH2 specific cytokines but also the expression of Ifng in stimulated naïve T cells (Williams et al. 2010). Mahmoudi and colleagues (2002) showed that the GAGA can bridge two DNA elements on separate chromosome by forming a protein link. Bridging by GAGA is dependent on its oligomerization POZ domain. There are numerous other examples of trans interaction in the literature and possibly more of such evidences will emerge in the future and add another layer of complexity in transcriptional regulation mediated by enhancer-promoter interaction. One of the hypotheses tested in this work is whether there is interchromosomal association or bridging between UASGAL sites located on different chromosomes mediated by the interaction between Gal80 molecules bound to different UASGAL-bound Gal4.

1.2.4 Activators: Activators are the trans-acting protein factors that bind to cis- acting elements to regulate transcription. As the name suggests, activators bind to cis-acting elements like enhancers and activate transcription. Eukaryotic transcription activators are modular proteins that are typically composed of a sequence specific DNA-binding domain and an activation domain. Through their activation domain they interact with the RNAP transcription apparatus. It may also modify or alter the structure. Based on the amino acid composition, 11 activation domains are classified as acidic (viral VP16 and yeast Gal4), glutamine- rich (SP1) or proline-rich (CTF). Activators are usually categorized by their DNA- binding domains. There are several well-characterized DNA-binding domains including C2H2 zinc-finger, Zn(II)6Cys2 binuclear cluster, homeo box, helix-turn- helix, bZIP, forkhead, ETS, POU etc. For many genes in metazoans, multiple activators bind to a single enhancer to exert a combinatorial effect on the regulation of transcription enabling a differential regulation of many genes with a relatively small number of transcriptional activators (Ptashne and Gann 2002).

The activators themselves are regulated in several ways. One way is through controlling the subcellular localization of the activators. For example, in the absence of insulin and growth factors, FOXO transcription factors are localized in the nucleus where they upregulate the expression of genes that are associated with arrest, stress resistance and cell death (Greer and Brunet 2005 and references therein). In the presence of insulin and growth factors, FOXO transcription factors are phosphorylated by Akt (also known as protein B) and SGK (serum and glucocorticoid inducible kinase). This leads to rapid re-localization of FOXO transcription factors from the nucleus to the cytoplasm (Greer and Brunet 2005 and references therein). Sequestration of FOXO transcription factors in the cytoplasm results in inhibition of FOXO-dependent transcription thereby allowing cell proliferation, stress sensitivity, and cell survival (Greer and Brunet 2005 and references therein). Additionally, the activators are regulated by masking the activation domain as in the case of Gal4 by Gal80 (Torchia et al. 1984, Yocum and 12 Johnston 1984, Giniger et al. 1985, Lohr and Hopper 1985, Johnston et al. 1987, Lue et al. 1987, Ma and Ptashne 1987a, Selleck and Majors 1987, Chasman and Kornberg

1990, Leuther and Johnston 1992, Mizutani and Tanaka 2003, Ptashne and Gann

2002). Yet another way is to degrade the activator after each round of activation as with Gcn4 and possibly Gal4 as well (Chi et al. 2001, Muratani et al. 2005).

One of the most important features of eukaryotic DNA is that it is packaged into , which affects all stages of transcription from activator binding and PIC

(pre-initiation complex) formation to elongation (reviewed in Workman and

Kingston 1998). The is the fundamental unit of chromatin. It is composed of an octamer of the four core (H2A, H2B, H3, and H4) around which 147 base pairs of DNA are wrapped. Chromatin structure primarily acts as a barrier to access of the DNA template. Eukaryotic transcriptional activators like Gal4 need to function despite the that the DNA is packaged into nucleosome. For example, nucleosomes in the GAL1 and GAL10 promoters must be removed or altered to allow formation of transcriptional complex. Several experiments showed that nucleosomes present in the inactive promoters of GAL1 and GAL10 are removed upon activation in a Gal4-dependent fashion (Fedor and Kornberg 1989,

Lohr et al.1987, Cavalli and Thoma 1993, Li and Smerdon 2002, Lee et al. 2004, Lohr

1997, Schwabish and Struhl 2004). Understandably, one critical function of activators is to recruit a host of coactivators that modify chromatin structure. Some of the well known chromatin-modifiers are ATP-dependent complexes such as Swi/Snf (Clapier and Cairns 2009 and references therein), 13 complexes with tail modifying activities such as yeast SAGA complex which contains the histone acetyltransferases Gcn5 that acetylates histones (Brownell et al. 1996,Grant et al. 1997, Li et al. 2007), PCAF (the mammalian SAGA complex) etc.

(Lee and Workman 2007). In addition to these chromatin-modifiers, activators also recruit , which acts as an interface, integrating and transducing regulatory information from the enhancer-bound activators to the basal transcription apparatus and RNA PolII (Myers and Kornberg 2000). The core yeast mediator is a

21-subunit complex, which was originally identified as a requirement for the activator-dependent stimulation of RNA polymerase II transcription (Bjorklund and

Gustafsson 2005 and references therein). Structure and function of the mediator seem to be conserved from yeast to humans (Bourbon et al. 2004).

To summarize, we observe that the eukaryotic transcriptional activators like Gal4 bind to specific DNA sequences that are located a great distance away from the promoters of the genes they regulate. Upon binding to these promoter-distal sequences (UASGAL for Gal4 in yeast, enhancers for metazoans,) they regulate transcription of genes by recruiting host of coactivators to alter or modify the chromatin structure of the promoters and facilitate the assembly of PIC. Such regulation from a distance is accomplished by protein-protein interactions mediated most likely by DNA looping that brings proteins bound at distant DNA sites in close contact. In fact, DNA looping seems to play a pivotal role in the transcriptional regulation both in the prokaryotes (including the transcriptional gene regulation of

14 bacteriophage λ) and the eukaryotes. Additionally, we also observe that long-range inter and intrachromosomal interaction mediated by protein-protein interaction is increasingly found to be important in coordinated gene regulation in eukaryotes. It will be interesting to know if such long-range interactions are involved in the transcriptional regulation of the GAL genes as well.

1.3. Genetic regulatory network of yeast galactose pathway:

The GAL/MEL system of genetic regulatory network is a galactose-responsive genetic switch in Saccharomyces cerevisiae. The striking feature of the GAL/MEL genetic switch is the tight repression of certain of the GAL genes in the absence of galactose and rapid and very high level of expression in the presence of galactose.

GAL genes encode enzymes, which are involved in utilization of galactose as the sole carbon source in the media. The GAL/MEL genetic switch in the budding yeast

Saccharomyces cerevisiae is one of the most extensively studied and well- characterized systems of transcriptional regulation. At the heart of this regulatory system are three proteins: Gal4 – the transcriptional activator, Gal80 – the inhibitor of Gal4 activity, and Gal3 – the sensor and transducer of galactose signaling. The transcriptional activator Gal4 binds to UASGAL sequences of the GAL genes both in the absence and presence of galactose. However, in the absence of galactose, Gal4 activity is blocked by the inhibitor Gal80, which interacts directly with the Gal4 activation domain (AD) and masks it from interacting with other components of 15 transcription machinery. In the presence of galactose, Gal3 interacts with Gal80 and relieves the Gal80 inhibition of Gal4 activity.

1.3.1 Enzymes for galactose utilization:

Galactose is converted to glucose-6-phosphate by the enzymes of Leloir pathway

(Johnston 1987 and references therein). These enzymes are encoded by GAL1

(kinase), GAL7 (), GAL10 (epimerase), and GAL5 (mutase) (Johnston

1987 and references therein). In addition, galactose enters the cells through the action of the permease encoded by GAL2, which is also under GAL regulation

(Johnston 1987 and references therein). Apart from the genes mentioned above,

MEL1, which encodes for α-galactosidase that breaks disaccharide melibiose into galactose and glucose is also under GAL regulation (Johnston 1987 and references therein).

Regulation of the expression of the GAL/MEL genes results in three states. a. Repressed state: In the presence of glucose, the expressions of most

GAL/MEL genes are severely repressed as cells utilize the preferred carbon source glucose for carbon and energy. b. Non-induced state: In the presence of carbon sources other than either glucose or galactose such as glycerol and lactic acid (glycerol + lactic acid) or raffinose, the expressions of most GAL genes are repressed and kept at low, basal level but poised for activation. The GAL regulatory system exerts variable level of 16 repression on different GAL genes allowing basal expression for some (e.g. GAL3 and GAL80) while almost no expression for others (GAL1, GAL10, GAL7, and GAL2). c. Induced state: In the presence of galactose, the GAL gene expression is induced rapidly and for some genes like the GAL1, 10, 7, and 2 to a very high level to almost 1000 fold induction (Johnston 1987 and references therein).

As mentioned previously, the three main regulatory players in the GAL genetic switch are Gal4, Gal80 and Gal3. Here a brief description of each will be given highlighting key elements that contribute to proper functioning of this genetic switch.

1.3.2 Gal4, the transcriptional activator: Gal4 is perhaps the most extensively studied transcriptional activator. It is an 881-amino acid (aa) (97 kDa) protein and a member of the Zn(II)2Cys6 binculear cluster family of proteins (McPherson et al.

2006). It exhibits a modular structure in which the DNA-binding, dimerization and nuclear localization functions reside within the N-terminal 1-147 amino acids

(Figure 1.1), and the transcription activation function is associated with two regions: the activation region 1 (AR1) which spans aa 148-225 and the activation region 2 (AR2) located on the C terminus aa 768-881. The function of the large middle region, which encompasses nearly two-thirds of the entire Gal4 protein, is not clear, although some studies indicate that it may have both positive and negative regulatory roles in Gal4 function.

17 Gal4 binds to several 17-bp sequences upstream of GAL genes termed as the UASGAL

(Guarente et al. 1982) with the palindromic motif 5’-CGG(N11)CCG-3’. The x-ray crystal structure of the DNA binding and dimerization domain of Gal4 bound to its cognate DNA site shows that Gal4 binds the DNA as a dimer (Hong et al. 2008). The

DNA-binding domain from the two subunits interact with the major groove of the

DNA on the opposite sides of the molecule (Hong et al. 2008).

The major transcriptional activation function is associated with AR2. AR2, which will be referred to as Gal4AD (or simply AD) also contains a region of aa 856-871 that is required for its interaction with Gal80. One feature of the activation region of

Gal4 is the preponderance of acidic amino acids similar to other well-known transcriptional activators like Gcn4 from yeast and VP16 protein from . The acidic nature of this domain appears to be important for its function (Gill et al. 1990); however, this is somewhat controversial as it was found that the hydrophobic residues within the activation domain rather than the acidic amino acids that are crucial for its function (Ruden 1992). Upon induction, the

Gal4AD interacts with various components of transcription machineries (Brent and

Ptashne 1985, Ma and Ptashne 1987). Notable amongst these are TBP (TATA- binding protein) and TFIIB (Transcription factor IIB) (Ansari et al. 1998, Wu et al

1996), Gal11 and Srb4 components of the mediator complex (Jeong et al. 2001;

Reeves and Hahn 2005, Koh et al. 1998), Tra1 subunit of the SAGA complex

(Bhaumik et al. 2004), Swi/Snf, Srb10, 19s proteasomal subunit Sug1 and Sug2 etc.

(Melcher and Johnston 1995, Yudkovsky et al. 1999, Hirst et al. 1999, Gonzalez et al. 18 2002). This clearly demonstrates that active Gal4 interacts with a large number of transcription factors and co-regulators to facilitate recruitment of RNAP and formation of PIC. Precise order and significance of these interactions are still subjects of active research. Employing a modified ChIP protocol, Bryant and

Ptashne (2003) showed that the SAGA and the mediator interact independently with

Gal4 upon induction and are recruited prior to RNAP recruitment. SAGA is found at

UASGAL1-10 as early as 4-7 minutes after galactose addition and may be required for recruitment of RNAP (Bryant and Ptashne 2003).

The function of the large middle region, which is almost two-thirds of the whole

Gal4 protein, is not clear. This region shares homology with a number of other transcriptional factors found in fungi (Poch 1997). Deletion analysis indicates that it has an inhibitory role in transcriptional activation (Stone and Sadowski 1993).

There is also evidence that it may have a secondary Gal80 (Sil et al.

1999). Moreover, a genetic selection for missense mutations that inactivate Gal4 found four single missense mutations within this region (Johnston and Dover 1988).

Somewhat contrary to these evidences, Gal4 lacking the entire middle region (mini

Gal4) retains the ability to activate transcription in a galactose dependent manner

(Ding and Johnston 1997) although the extent of activation is only up to 50% of that of a full-length Gal4. It is possible that this region acts as a flexible spacer between the Gal4 DBD and Gal4AD to allow UAS-bound Gal4 to interact and recruit various components of transcription machineries to GAL gene promoters.

19 Gal4 undergoes posttranslational modifications in the form of and ubiquitination, which may have regulatory roles in Gal4 activity. Gal4 migrates as three distinct species via SDS PAGE indicating three different phosphorylation states

(Gal4a, Gal4b, Gal4c, or Gal4I, II or III) (Mylin et al. 1989 Mylin et al. 1990, Sadowski et al. 1991). Under repressed or uninduced conditions, Gal4 shows only form “a” and

“b”. Gal4c or Gal4III is only detectable under the inducing condition and corresponds to the transcriptionally active form of Gal4 (Muratani et al. 2005, Mylin et al. 1989, Mylin et al. 1990, Sadowski et al. 1991). Amongst several phosphorylation sites in Gal4, S699 seems to be particularly important for activation function (Hirst et al. 1999). The Srb10 subunit of the mediator complex seems to be responsible for phosphorylation of S699 (Hirst et al. 1999) suggesting this to be an early event in galactose induction. Gal4 also undergoes ubiquitination and proteasomal degradation. Two different F-box Grr1 and Dsg1 have been implicated for ubiquitination and subsequent proteolysis of Gal4. Grr1 seems to be responsible for Gal4 turnover under repressing condition and Dsg1 seems to act on active Gal4 (Muratani et al. 2005). Residues important for polyubiquitination are not known. In addition to polyubiquitination mediated by

Grr1 and Dsg1, Gal4 also appears to undergo mono-ubiquitination by some as yet unknown E3 . Mono ubiquitination has been reported to protect Gal4 from destabilization by proteasomal ATPases (Ferdous et al. 2001). It seems that poly and mono ubiquitination have opposite effects on the stability of the Gal4-UAS interaction, as polyubiquitination targets Gal4 for protesomal degradation thereby destabilizing the Gal4-UAS complex. On the other hand, monoubiquitination 20 protects Gal4 from the destabilizing effect of proteasomal comoponents presumably

Sug1 and Sug2 (Muratani 2005, Ferdous 2001, Gonzalez 2002). It is not entirely clear what the significance is of these seemingly opposite effects. One possibility is that by keeping the active Gal4 for a brief period on UAS, the cell imposes the requirement for continuous galactose signaling to mediate activation thereby preventing unbridled transcription from activated UAS-bound Gal4 which could have a high energy cost. Sorting out precisely what the various modifications of Gal4 achieve singly or in combination will require much future work.

1.3.3 Gal80 – the transcriptional inhibitor:

Gal80 is a 435 aa (48 kDa) protein that acts as an inhibitor of transcription of the

GAL genes. In the absence of galactose, it binds and masks the Gal4AD from interacting with co-activators like SAGA (Lue et al. 1987, Carrozza et al. 2002). As mentioned previously, a region within the Gal4AD (aa 851-874) is required for interaction with Gal80 (Nogi et al. 1977, Johnston et al. 1987, Ma and Ptashne 1987).

This interaction was shown to be of high affinity in vitro (Kd ~2x10-8M~3X10-10M)

(Lue et al. 1987, Wu et al. 1996, Melcher and Xu 2001). In the presence of galactose, the galactose sensor Gal3 interacts with Gal80 and relieves Gal80 repression of Gal4 activity. Gal80 crystal structures both in complex with the Gal4AD and with Gal3 have been resolved (Kumar et al. 2008, Lavy et al. 2012). Gal80 complexed with the

Gal4AD showed that Gal80 is also bound to an NAD ligand at a specific pocket near the Gal4AD binding site (Kumar et al. 2008). Moreover, it was shown in this study that in-vitro NADP binding competes with the Gal80-Gal4 interaction. It would be 21 interesting to see if NADP binding has any physiological relevance with respect to

Gal80 function.

Another important feature of Gal80 is that it dimerizes with very high affinity (Kd

~1-3 x10-10 M) and also transiently tetramerizes with moderate affinity (Kd~5 x10-

8M) (Melcher and Xu 2001). Most importantly, this Gal80 dimer-dimer interaction may be critical for tight repression (Melcher and Xu 2001). The dimer of Gal80 is very dynamic with a very short half- of approximately 30 seconds, which perhaps explains why it migrates as a monomer on gel filtration columns (Melcher and Xu

2001, Yun et al. 1991, Timson et al. 2002). In-vitro binding assay showed Gal4AD-

Gal80 form a complex with a 2:2 stoichiometry (Melcher and Xu 2001). A reverse yeast two-hybrid selection for Gal80 mutants impaired in interaction with Gal3 showed that all the Gal3 non binder mutants that fail to self associate are also impaired in binding to Gal4AD (Pilauri et al. 2005). Based on these results it was proposed that self-association of Gal80 may be required for interaction with and inhibition of Gal4; however the exact oligomeric form of Gal80 that inhibits Gal4 activity in vivo is yet to be determined. The stoichiometry of the Gal80-Gal3 complex is not clear. The crystal structure of Gal80 with Gal3 showed a 2:2 heterotetramer where two monomers of Gal3 interact with a dimer of Gal80 (Lavy et al. 2012).

However, gel filtration analysis of Gal80-Gal3 complex indicated it to be a heterodimer (Timson et al. 2002). It is to be noted that although the crystal structure showed a 2:2 heterotetramer of Gal3-Gal80, this may be the most stable structure under crystallization condition and not necessarily the predominant 22 species in vivo. Taken together, these evidences barring perhaps the stoichiometry of the Gal80-Gal3 crystal structure suggest that disruption of Gal80 self-assemblies by Gal3 may be the mechanism for adversely affecting Gal80’s ability to bind and inhibit Gal4.

1.3.4 Gal3 – the galactose sensor and signal transducer:

Gal3 mediates the galactose signal response. It is a 520 aa protein with 74% sequence identify with another GAL gene galactokinse or Gal1. Unlike Gal1, Gal3 does not have kinase activity. Gal3 directly interacts with Gal80 in the presence of galactose and ATP (Suzuki-Fujimoto et al. 1996; Zenke et al. 1996) and destabilizes the Gal80-Gal4AD interaction (Sil et al. 1999).

Like Gal1, Gal3 is a member of the family of small metabolite that includes the galactose kinases, homoserine kinases, mevalonate kinases, and phosphomevalonate kinases (GHMP kinase family) (Bork et al. 1993). Like other kinases in the family, Gal3 has a phosphate-binding pocket. Binding of ATP in this pocket appears to increase galactose-mediated interaction between Gal3 and Gal80

(Lavy et al. 2012).

1.3.5 The current model of the GAL gene switch: According to current understanding of this genetic switch, the transcriptional activator Gal4 binds to specific sites in the promoter termed as UASGAL sites as a dimer both in the absence and presence of galactose. In the absence of galactose, the Gal4AD is masked by the 23 interaction with the transcriptional inhibitor Gal80 as depicted in Figure 1.2 (left) keeping Gal4 inactive. In the presence of galactose, the signal transducer Gal3 interacts with Gal80 relieving Gal80 inhibition of Gal4 activity. This allows Gal4 to recruit RNAP to the GAL gene promoters through interactions with different coactivators like SAGA, mediator etc. and thus activate the transcription of the GAL genes (shown in Figure 1.2 right).

24

Figure 1.1

10 51 DNA Binding 148 196 768 881 Transcription activation 856 881 Gal80 Interaction

1 74 74 Nuclear Transport

1 881

FIGURE 1.1: Functional domains of Gal4 protein. (Adapted from Johnston 1987; used with permission)

25

Figure 1.2.

Figure 1.2: Model of the GAL gene switch at the time of initiation of this thesis work. Left: in the absence of galactose Gal80 interacts with Gal4AD and masks Gal4 activity thus keeping the gene expression “OFF” and in the presence of galactose, Gal3 binds to Gal80 thereby relieving Gal80 inhibition of Gal4. Right: Ga4AD free of Gal80 interacts with various coactivators like the SAGA, mediators etc. to recruit RNAP complex and activate the transcription of GAL genes.

(From Prof. Jim Hopper of The Ohio State University,

used with permission)

26

CHAPTER 2:

Interaction of Gal3 with Gal80 impairs Gal80

self-association

This work is part of the following publication

Egriboz O, Goswami S, Tao X, Dotts K, Schaeffer C, Pilauri V, Hopper JE. 2013 Self- association of the Gal4 inhibitor protein Gal80 is impaired by Gal3: evidence for a new mechanism in the GAL gene switch Mol Cell Biol.; 33(18):3667-7

27

2.1 ABSTRACT:

The DNA-binding transcriptional activator Gal4 with its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to upstream activation sequences or UASGAL sites in GAL gene promoters as a dimer both in the absence and presence of galactose. In the absence of galactose a Gal80 dimer binds to and masks the Gal4 activation domain, inhibiting its activity. In the presence of galactose, Gal3 interacts with Gal80 and relieves

Gal80’s inhibition of Gal4 activity allowing rapid induction of expression of GAL genes. The mechanism of how exactly Gal3 affects Gal80 to relieve inhibition has been an unresolved question. Previous analyses of Gal80 mutants led to the idea of competition between Gal3-Gal80 with Gal80 self-association interactions. In this body of work, in-vitro chemical cross-linking coupled with SDS-PAGE was employed along with native PAGE analyses as well as live cell fluorescence microscopy to ascertain the effect of Gal3 on Gal80 self association. These analyses revealed that

Gal80 self-association is impaired in the presence of Gal3 that can interact with

Gal80 suggesting Gal3-Gal80 interaction negatively affects Gal80 self association.

28 2.2 INTRODUCTION:

Regulation of the activity of transcription activators is a common strategy eukaryotes employ to modulate gene expression. Transcription factors are activated or inhibited by signaling processes in a variety of ways, including through ligand binding, protein-protein interactions and chemical modifications (Bannister et al.

2000, Calkhoven et al. 1996, Desterro et al. 2000, Kumar et al. 2008, Lenburg et al.

1996, Reece et al. 1997, Whitmarsh et al. 2000). Direct masking of the activation domain (AD) of a transcriptional activator by an inhibitory protein and relief of such masking in response to signal is typical for several eukaryotes (Carman et al. 2007,

Dyson et al. 1998, Lue et al. 1987, Ma and Ptashne 1987, Marine 2007). For example, the transcriptional inhibitors Rb, MDM4/MDMX, ZFM1, Opi1, and Gal80 interact directly with DNA binding transcriptional activators to exert their inhibitory effects

(Carman and Henry 2007, Dyson et al. 1998, Marine et al. 2007, Zhang et al. 1998).

Gal4, the DNA binding transcriptional activator of the GAL gene switch that controls expression of the galactose pathway genes in Saccharomyces cerevisae is inhibited in the absence of galactose by Gal80 through Gal4-Gal80 interaction that masks Gal4 activation domain (AD) (Carlson M 1987, Johnston M. 1987, Johnston and Carlson

1992, Johnston and Hopper 1982, Lohr et al. 1995, Rubio-Texeira M. 2005). In the presence of galactose, Gal3 interacts with Gal80 and relieves Gal80 inhibition of

Gal4 activity. Several studies have established that Gal3-Gal80 complex formation is required for the relief of Gal80 inhibition of Gal4AD and Gal4-mediated transcription activation of the GAL genes (Bhat and Hopper 1992, Blank TE 1997, 29 Vollenbroich 1999, Yano and Fukasawa 1997). This leads to rapid induction with readily detectable GAL mRNA within 5-10 minutes of galactose exposure (Bryant and Ptashne 2003, St. John and Davis 1981, Yarger et al. 1984).

Exactly how galactose-activated Gal3 binding to Gal80 alters Gal80 to overcome inhibition of Gal4 was not clear prior to this work. One hypothesis involves a simple competition between Gal4 and Gal3 for binding to Gal80 similar to what has been proposed for the somewhat similar GAL gene switch of the distantly related yeast

Kluyveromyces lactis (K lactis)(Rubio-Texeira M. 2005, Anders A et al. 2006,

Salmeron and Johnston 1986, Webster and Dickson 1988.). In this system, the binding of K. lactis Gal1 (KIGal1-performs functions similar to ScGal3 in K. lactis with respect to the GAL gene switch) to KIGal80 overcomes KIGal80 inhibition of KIGal4 activity (Anders A et al. 2006, Zenke et al. 1996). The experimental evidence supports the view that KIGal1 and KIGal4 binding to KIGal80 are mutually exclusive and that a heterotetrameric KIgal80 dimer-KI Gal1 dimer complex forms in response to galactose. Based on mathematical modeling of that system, it was suggested that two KIGal1 monomers somehow compete with KIgal80-KIGal4 dimer-dimer interactions (Anders et al. 2006). A similar mechanism may be in place in the S. cerevisiae GAL gene switch as well. Overexpression of either Gal4AD or Gal3 relieves Gal80 inhibition of Gal4 in the absence of galactose (Johnston et al. 1986,

Ma and Ptashne 1987, Johnston and Carlson 1992, Blank et al 1997, Hashimoto et al

1983) and increasing Gal80 concentration reverses the effect (Nogi et al. 1984), which is consistent with the above-mentioned hypothesis. However, there has been 30 no direct physical evidence of such simple competition taking place in the S. cerevisiae GAL gene switch. On the contrary, analyses of the Gal3-Gal80-galactose-

ATP structure (Lavy et al 2012) together with the Gal80-Gal4AD structure from S. cerevisiae (Kumar et al. 2008) and K. lactis (Thoden et al. 2008) led Lavy and coworkers (2012) to suggest that Gal3 and Gal4AD do not compete for the same surface on Gal80.

In this work, in-vitro chemical crosslinking coupled with SDS-PAGE was employed in addition to fluorescence imaging of live cells using spinning disk confocal microscopy to study the effect of Gal3 on Gal80 self assemblies. It was consistently observed that Gal80 self-association is reduced in the presence of Gal3 that can interact with Gal80. Gal3-mediated decrease in Gal80 multimers may represent the mechanistic event leading to the relief of inhibition of Gal4. This proposal is consistent with the observed correlation between the Gal80 self-association interactions and the inhibition of Gal4 (Melcher and Xu 2001.).

2.3 MATERIALS AND METHODS:

2.3.1 Yeast strains and plasmids: S. cerevisiae strain Sc857 (MATa ade1 ile leu2-

S-2 3,112 ura3-52 trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1-HIS3 Gal80 -

2mCitrine-KAN) was used to study the effect of galactose on Gal80S-2 cluster. S. cerevisiae strain Sc858 (MATa ade1 ile leu2-3,112 ura3-52 trp1-HIII his3-Δ1 MEL1

LYS2::GAL1UAS-GAL1-HIS3 gal3 Δ ::LEU2 Gal80-2mCitrine-KAN) was used for 31 ascertaining the effects of various Gal3 mutants on dissipation of Gal80-2mCitrine clusters.

The plasmids used in this study are provided in Table 2.1.

Table 2.1

Plasmid Relevant genotype Reference or Source pCLAKS82 6XHis-Gal80 KANr Egriboz et al . 2013 pAKS118 6XHis- Gal80S-2 KANr Egriboz et al . 2013 pAKS123E116G 6XHis-Gal3-E116G KANr Egriboz et al . 2013 pTEB16 CEN ARS1 TRP1 PGal3-Gal3 Blank et al. 1997 pTEB16D368V CEN ARS1 TRP1 PGal3-Gal3-D368V Blank et al. 1997 pTEB16D111C CEN ARS1 TRP1 PGal3-Gal3-D111C Diep et al. 2008 pRS414 CEN ARS1 TRP1 New England Biolabs

Plasmids were constructed using standard molecular cloning and PCR techniques.

2.3.2 Microscopy:

All microscopy experiments were carried out with a Nikon TE-2000U spinning disk confocal microscope that was equipped with a 100X/1.4 –numerical aperture (NA) objective lens (Nikon, Melville, NY), 488-, 514-, and 568-nm argon ion lasers, and a charge-coupled device camera (ORCA-AG; Hamamatsu, Bridgewater, NJ). The effect

32 of galactose on Gal80S-2-2mCitrine was studied using the following protocol: Cells

(Sc857) were grown to mid-log phase in 3% glycerol 2% lactic acid synthetic complete media and immobilized in the Y04C microfluidics plates (CellASIC,

Hayward, CA). Images were acquired before and after addition of galactose to the medium circulated in the cell chamber. Effects of different Gal3 mutants on dissipation of WT Gal80-2mCitrine clusters were carried out as follows: Cells (Sc858

– transformed with either the mutant or the WT Gal3 expressed from its own promoter in CEN vector) were grown to early-mid log phase and then induced with galactose for one hour. Cells were immobilized on glass slides using 10% gelatin before image acquisition.

2.3.3 Protein expression and purification:

Affinity purification was used to purify 6×His-tagged versions of Gal3 and Gal80.

6×His-Gal80 from pCLAKS82 and 6×His-Gal3 from pAKS123 were expressed in the E. coli Rosetta (DE3) strain using standard protocols. The cells were resuspended in 45 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole [pH 8.0]) with one tablet of protease inhibitor cocktail (Roche) and were lysed in a French press with a pressure of 1,200 lb/in2. Purified 6×His-tagged proteins were pulled down with Talon beads according to the manufacturer's protocol (Clontech). They were then buffer exchanged into a buffer composed of 15 mM KH2PO4, 150 mM KCl, 10% glycerol, 2 mM DTT, and 1 mM PMSF. Proteins were

33 then concentrated using the Amicon Ultra-4 system (catalog no. UFC800308; EMD

Millipore, Billerica, MA).

2.3.4 Cross-linking of Gal80 and Gal3.

The cross-linking reaction was conducted as described elsewhere (Adams et al.

2009). Briefly, 2μM Gal80 were incubated with Gal3 at the concentrations indicated in the figures in the presence of 500 μM ATP and 25 mM galactose in a total reaction volume of 30 μl at 4°C for 1 hour. Formaldehyde was then added to the samples to a final concentration of 30 mM, and the samples were incubated at 4°C for an additional 2 hours to cross-link the proteins. A volume of 10 μl of 4× SDS electrophoresis loading buffer was added to each sample, and the samples were incubated at room temperature for additional 10 minutes. Proteins were separated by 7.5% SDS-PAGE and visualized by coomassie blue staining.

2.3.5 Discontinuous blue native protein gel electrophoresis:

To check whether 6×His-Gal80S-2has a higher propensity to oligomerize compared to 6×His-Gal80, native gel electrophoresis was conducted as described previously

(Niepmann and Zheng J 2006) that allows the separation of proteins according to their size, oligomeric state, and shape. Different amounts of purified 6x His-tagged

Gal80 and 6xHis-tagged Gal80S-2 mixed with gel loading buffer (100 mM Tris-Cl [pH

8.0], 40% glycerol, 0.5% Serva Blue G) and incubated for 10 min at room temperature. The protein species in the samples were analyzed on nondenaturing 34 polyacrylamide precast 4-20% blue native (BN) -gradient gel from Jule Inc. (Milford,

CT, USA). Histidine (final concentration of 100 mM; pH 8.0) and 0.002% Serva Blue

G were added to the cathode buffer prior to electrophoresis. Catalase (230 kDa), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (143 kDa), and bovine serum albumin (BSA) (69 kDa) were run as molecular markers. The gels were destained with several changes of 7.5% acetic acid/5% ethanol.

2.4 RESULTS:

2.4.1 Gal80-Gal80 self-association is impaired under conditions supporting

Gal3-Gal80 interaction: Previously it was shown that in-vitro Gal80 dimerizes very strongly (Kd of ~ 0.1-0.3 nM) and transiently tetramerizes with moderate affinity

(Kd ~ 50 nM) (Melcher and Xu 2001.). In-vitro chemical crosslinking coupled with

SDS-PAGE was used to ascertain if Gal3 affects Gal80 self-association. In the absence of Gal3, multiple bands could be detected corresponding to crosslinked Gal80 molecules (Lane 4, Figure 2.1A) confirming oligomerization of Gal80 in-vitro. The mobility of the most prominent cross-linked Gal80 species would suggest it to be a dimer of Gal80, which is consistent with earlier findings that Gal80 dimerizes with very high affinity (Melcher and Xu 2001). However, it may also be that capturing higher order oligomers would require multiple cross linking events compared to just one for detection of dimers. Therefore, the bands may not necessarily reflect the abundance of different native oligomeric states of Gal80. No additional crosslinked band for purified 6x-His tagged Gal3 alone was detected (Lane 2, Figure 2.1A). In the presence of 6x-His tagged Gal3 with ATP and galactose, it was observed that there 35 was a significant decrease in the cross-linked Gal80 species (Lane 5, Figure 2.1A). To address if the disappearance of cross-linked Gal80 species is due to interaction between Gal3 and Gal80, a mutant Gal3 (Gal3E116G) that does not bind Gal80 (Diep et al 2008) was used in place of WT Gal3 and the same cross-linking experiment was carried out as described above. The cross-linked Gal80 species were not diminished in the presence of mutant Gal3 (Figure 2.1B Lane 5) suggesting that it is not galactose by itself but rather Gal3-Gal80 interaction that is required to diminish cross-linked oligomeric Gal80. To further establish that Gal3-Gal80 interaction is required to reduce cross-linked Gal80 multimers, the same experiment was repeated but this time in the presence and absence of ATP and galactose since Gal3 interacts strongly with Gal80 only in the presence of ATP and galactose (Bajwa et al.

1988, Bhat and Hopper 1992, Suzuki-Fujimoto et al. 1996, Blank et al. 1997, Yano and Fukusawa 1997, Platt and Reece 1998, Peng and Hopper 2002, Pilauri et al.

2005). In the presence of ATP and galactose, cross-linked Gal80 species showed a gradual decrease in abundance, but in the absence of ATP and galactose this effect of

Gal3 was significantly reduced even when very high concentration of Gal3 (18μM) was used (Figure 2.2A lanes 12 and 13). As expected, mutant Gal3E116G impaired in binding to Gal80 had very little effect on the Gal80 cross-linking and showed minimal effect at a very high concentration and even that effect also was reduced in the absence of ATP and galactose (Figure 2.2B). These two results collectively show that Gal3 impairs Gal80 self-association only when it has the capacity to bind to

Gal80.

36 2.4.2 The nuclei of live cells display clusters of Gal80-2mYFP that dissipate in the response to galactose-triggered Gal3-Gal80 interaction:

Using spinning disk confocal imaging, it was discovered that Gal80-2mYFP can be seen as intranuclear clusters when cells are grown in glycerol-lactic acid media and these clusters dissipate within 30 minutes after galactose addition (Egriboz et al.

2013). To ascertain if the galactose-triggered Gal3-Gal80 interaction is required for the dissipation of these intranuclear Gal80-2mYFP clusters, live cell imaging was carried out. Gal80S-2 a super repressor, non-inducible variant of Gal80 that is impaired in binding to Gal3 was used to observe if it exhibits such dissipation of clusters in the presence of galactose. Even after 1 hour in galactose, the Gal80S-2-

2mYFP clusters did not show any dissipation suggesting Gal3 binding to Gal80 is necessary for dissipation (Figure 2.3A). To further confirm the requirement of Gal3-

Gal80 interaction for dissipation of Gal80-2mYFP clusters, previously characterized

Gal3 mutants (Blank et al. 1997, Diep et al 2008) were utilized. Either WT Gal3 or different mutant versions of Gal3 were expressed from GAL3 promoter in a low copy CEN vector in the yeast strain Sc858 (Δgal3) and were grown either in the non- inducing 3%glycerol-2%lactic media or in the inducing 3%glycerol-2%lactic acid with 2% galactose media. In the presence of WT Gal3, Gal80-2mYFP showed clusters only when grown in the non-inducing glycerol-lactic media but not in the inducing media containing galactose in addition to glycerol-lactic acid (Figure 2.3B).

In the absence of Gal3 (empty vector) clusters were visible both in the absence of galactose (glycerol-lactic acid media) and in the presence of galactose (glycerol- lactic acid with galactose) (Figure 2.3C). Furthermore, when a mutant of Gal3, 37 Gal3D111C, which is impaired in binding to Gal80 (Diep et al. 2008), was used, clusters were observed even 1 hour after galactose addition (Figure 2.3D). On the other hand, when Gal3c-D368V, a mutant of Gal3 that can interact with Gal80 even in the absence of galactose (Blank et al. 1997), was expressed no Gal80-2mYFP clusters were visible either in the absence or in the presence of galactose (Figure

2.3E). Taken together, the results from the live cell imaging experiments indicate that the dissipation of Gal80-2mYFP clusters require Gal3-Gal80 interaction.

2.4.3 The super repressor Gal80S-2 forms more stable higher order oligomers on native gel compared to WT Gal80.

Native 4-12% polyacrylamide gradient was used to ascertain Gal3’s effect on Gal80 self-association, (Egriboz et al. 2013). Based on one experiment, it seemed that the presence of Gal3 (with ATP and galactose) leads to reduction in higher order oligomeric forms of WT Gal80 but not those of Gal80S-2 where the two forms of

Gal80 (WT and super repressor variant) were run on separate gels (Egriboz et al.

2013). Comparing the two gels, it appeared that Gal80S-2 made more prominent higher order oligomers than WT Gal80. To follow-up on this observation, both purified 6x His-tagged Gal80S-2 and WT Gal80 were run on the same precast 4-20%

BN-polyacrylamide gel (blue-native polyacrylamide gel). Consistently Gal80S-2 showed more prominent higher order oligomeric forms (Figure 2.4). This along with the in-vivo microscopic observation of nondissipation of Gal80S-2 -2mYFP seem to suggest that this mutant may form more stable oligomeric structures in-vivo and 38 such stable oligomeric forms may interfere with Gal3’s ability to disrupt the self assemblies.

2.5 DISCUSSION:

In this study, in-vitro cross-linking, native gradient gel along with live cell imaging using spinning disk confocal microscopy were used to study the effect of Gal3 binding to Gal80 on Gal80 self associations. Both lines of investigation indicate that

Gal3-Gal80 interaction substantially reduces Gal80 self-associations.

Both native and formaldehyde-treated samples of Gal80 showed multiple forms of

Gal80 with electrophoretic mobilities consistent with those of dimers and higher order oligomers. These forms of Gal80 were diminished in response to increases in concentration of Gal3 in the presence of galactose.

It should be noted that when purified untagged Gal3 and Gal80 were used in the crosslinking experiment in place of 6xHis-tagged proteins (Egriboz et al. 2013) it was observed that reduction of crosslinked oligomeric Gal80 species was accompanied by emergence of a new band whose mobility was consistent with that of a Gal3-Gal80 complex. This study did not reveal the emergence of any new complex in the crosslinking experiments with 6xHis-tagged Gal3 and Gal80. The purification strategy utilized for obtaining untagged Gal3 and Gal80 required Gal3-

Gal80 interactions rendering this strategy inapplicable for purification of mutants of 39 Gal3 and Gal80 that impair such interaction. So the apparent absence of crosslinked

Gal3-Gal80 complex in this study may be due to low crosslinking efficiency between the two tagged proteins. Another possibility is that the electrophoretic mobilities of the new 6xHis tagged Gal3-Gal80 complex may be similar to the mobility of a dimer of 6xHis-tagged Gal80 and therefore was not resolved. Nevertheless, consistently a reduction in 6xHis-tagged Gal80 oligomers was observed in the presence of Gal3 that can interact with Gal80 but not with the Gal3 mutant that cannot. Moreover, this effect of Gal3 was dependent on ATP/galactose, ruling out variation in crosslinking efficiency as a possible reason for disappearance of crosslinked Gal80 oligomers.

Live-cell imaging experiments demonstrate that the newly discovered Gal80-2mYFP clusters dissipate only under the condition favorable for Gal3-Gal80 interaction. One reasonable hypothesis about the Gal80-2mYFP clusters is that they are Gal80 self- association assemblies. One can further hypothesize that Gal3 interferes with the formation and/or stability of these self-assemblies. A more detailed analysis of these

Gal80 clusters will be the topic of the next chapter.

One important question regarding these observations is what the physiological relevance of Gal3’s effect on Gal80 self-association could be. In 2001, Melcher and

Xu showed that Gal80 dimer-dimer interactions may be required for tight repression of GAL genes (Melcher and Xu 2001.). Their studies provided a possible explanation for the difference in the basal level of expression between MEL1 and 40 GAL1. MEL1 contains just a single UASGAL site and shows significant Gal4-dependent basal expression in the absence of galactose whereas GAL1 with four UASGAL sites shows very little to no basal expression (Melcher and Xu 2001). Additionally, the x- ray crystal structures of dimeric and tetrameric forms of Gal80 and a Gal80-Gal4AD complex respectively from the Joshua-Tor lab support the notion that Gal80 and

Gal4 can interact to form a 2:2 heterotetramer (Kumar et al. 2008). This work also identified an amino acid substitution (N230R) within the Gal80 dimerization surface that impairs Gal80 self-association also impairs Gal80Gal4AD interaction and Gal80 inhibition of Gal4 transcriptional activity (Kumar et al. 2008). Taken together, the results from these two studies support the idea that Gal80 self-association plays a physiological role in the GAL gene switch. The work described in this chapter along with other experimental evidence as presented in the publication Egriboz et al.

(2013) provide a possible mechanism of how Gal3 alters Gal80 to bring about relief of Gal80 inhibition of Gal4 activity.

41

Figure 2.1. Gal3’s capacity to impair Gal80 self-association depends on its capacity to bind to Gal80. (A) 6xHis-Gal3 (9 μM) was incubated with 500 μM ATP and 25 mM galactose without cross-linker (lane 1) or with 30 mM formaldehyde

(lane 2). 6xHis-Gal80 (12 μM) migrated as a single band without the cross-linker at the expected mobility of the monomer (lane 3). Cross-linked 6xHis-Gal80 (with 30 mM formaldehyde in the presence of 500 μM ATP, 25 mM galactose at 4°C) formed higher mobility bands (lane 4). These higher mobility bands diminished in presence of 6xHis-Gal3 under the same conditions as above (lane 5). The samples were resolved on a 7.5% SDS polyacrylamide gel and subsequently stained with coomassie blue. (B) Same as in part (A) except the gal3 mutant (6xHis- Gal3E116G) defective for binding to Gal80 was used instead of 6xHis-Gal3.

42 Figure 2.1.

1. A

B.

43

Figure 2.2. Increasing levels of Gal3 binding reduced cross-linked Gal80 molecules in an ATP/Galactose dependent manner: Fixed amount of Gal80 was incubated with increasing amount of Gal3 (WT-2A and mutant gal3* -- gal3E116G in 2B) in the presence of 500 µM ATP, 25 mM galactose and 30 mM formaldehyde at

4°C (lanes 5-8). In lane 9 same amount of Gal3 (WT or mutant) was incubated with

12µM Gal80 without ATP or galactose. The samples were resolved on a 7.5% SDS- polyacrylamide gel, and were subsequently stained with coomassie blue. +XL –with crosslinking; -XL—without crosslinking

44

Figure 2.2. A.

Lane 1 2 3 4 5 6 7 8 9 ATP/Gal + + + + + + + + - Gal80 - - + + + + + + + GAL3WT(μM) 18 18 0 0 2 6 12 18 18

80(2) 80(2) – ATP/GAL

80(2) + ATP/GAL

Protein Gal3 Gal3 Gal80 Gal80 Standards -XL +XL -XL +XL + XL B.

Lane 1 2 3 4 5 6 7 8 9 ATP/Gal + + + + + + + + - Gal80 - - + + + + + + + Gal3*( μM) 15 15 0 0 2 6 9 12 12

80(2) 80(2) – ATP/GAL

80(2) + ATP/GAL

Protein Gal3* Gal3* Gal8 Gal80 Standards -XL +XL -XL +XL + XL

45

Figure 2.3. Gal3’s capacity to bind to Gal80 is required for galactose-triggered dissipation of nuclear Gal80 clusters. 3A. Sc857 cells expressing Gal80S-2-2mYFP were grown to mid-log phase in glycerol-lactic acid containing media and then images were acquired either in the absence of galactose or in the presence of galactose (1 hour post addition of galactose). 3B-3E, Sc858 (gal3Δ) cells were transformed with low copy CEN vectors expressing either (B) WT Gal3 (pTEB16); or

(C) no Gal3 (pRS414); or (D) a mutant of Gal3 (pTEB16-D111C) impaired in binding to Gal80; or (E) a mutant Gal3 (pTEB16-D368V) that is capable of binding to Gal80 independent of galactose. Arrows indicate Gal80-2mYFP clusters.

46 2.3A.

GALACTOSE

- +

Gal80S-2-2mYFP

DIC

B.

Gal80-2mYFP

DIC

continued…

47

…Figure 2.3 continued

C.

GALACTOSE

- +

Gal80-2mYFP

DIC

D.

Gal80-2mYFP

DIC

continued…

48 …Figure 2.3 continued

E.

GALACTOSE

- +

Gal80-2mYFP

DIC

49

Figure 2.4. The Gal80S-2 variant that is defective in binding to Gal3 shows more prominent multimerization than the WT Gal80. Equal amounts of Gal80 and Gal80S-2 (5, 10 and 15 μM) were run on a precast 4-20% Blue-Native gel.

Amounts of proteins loaded in each lane are indicated.

KD

250

Gal80(2)

150

69

Concn (μM) 15 10 5 5 10 15

6xHis-Gal80S-2 6xHis- Gal80 WT

50

CHAPTER 3:

Investigation Of The Intranuclear Clusters of Gal80 and The Intranuclear Foci Of Gal4

51 3.1 ABSTRACT:

Work from several labs including the J Hopper lab have established that Gal80 self associates and this self-association may be critical for its functioning. Recently a former graduate student in the J Hopper lab, Onur Egriboz discovered that Gal80-

2GFP/2mYFP exhibits intranuclear clusters that dissipate in the presence of galactose. In the work described in the previous chapter, it was observed that the galactose-triggered dissipation of Gal80 cluster is dependent on Gal3-Gal80 interaction. In this work, more extensive investigation into the nature of these Gal80 clusters was carried out. This work revealed that these clusters associate strongly with the UASGAL sites in the GAL1-10-7 locus. Moreover the occurrence of these

Gal80 clusters is dependent on the presence of Gal4. Additionally, it was discovered that Gal4-2GFP/2mYFP makes distinct intranuclear foci that associates strongly with the GAL1-10-7 locus, but in sharp contrast to Gal80 clusters, the Gal4 foci do not dissipate in response to galactose. This work shows that both Gal80 clusters and

Gal4 foci associate with the GAL1-10-7 locus and behave differently consistent with their function. However, association of what seems to be large molecular assemblies of Gal80 and Gal4 with the GAL1-10-7 locus raises interesting possibilities as to their mechanistic significance for the GAL gene regulation.

52

3.2 INTRODUCTION:

In chapter 2 it was mentioned that Gal80-2GFP/2mYFP makes intranuclear clusters when grown in non-inducing 3%glycerol-2%lactic acid media and that these clusters dissipate upon addition of galactose (Egriboz et al. 2013). Preliminary studies suggested that these cluster might represent large molecular assemblies of

Gal80 and that the dissipation of the clusters is dependent on the Gal3-Gal80 interaction (Egriboz et al. 2013). Given the importance of Gal80 self association in the GAL gene regulation (Bram et al. 1986, Melcher and Xu 2001, Kumar et al. 2008,

Egriboz et al. 2013) and occurrence of what appears to be large molecular self assemblies of Gal80, it became important to investigate these Gal80 clusters further to gain better understanding of their role in the GAL genetic switch.

This work has attempted to address two central questions regarding the Gal80 clusters. The first question is “what is the basis of formation of these Gal80 clusters?” The second question is “what is (if any) the functional significance of these clusters?” Spinning disk confocal microscopy was employed to address these two questions. It showed that Gal80 clusters associate strongly with the UASGAL sites in the GAL1-10-7 locus and also occurrence of Gal80 cluster requires the presence of

Gal4. Additionally, it was discovered that Gal4 when tagged with 2GFP or 2mYFP also makes visible intranuclear foci, which also strongly associates with the GAL1-

10-7 locus. Furthermore, the results obtained in the initial experiments suggest that association of these Gal80 clusters with UASGAL sites may lead to repression of the 53 GAL gene even in the presence of galactose. Additionally, employing a previously described fluorescence quantitation method (Coffman et al. 2011), number of molecules in the Gal80 clusters and Gal4 foci were estimated.

3.3 MATERIALS AND METHODS:

3.3.1 Yeast strains, plasmids, media and growth conditions: Yeast strains and plasmids used are listed in table 3.9 and 3.10 respectively. Yeast strains were grown at 30°C in standard nonselective YEP medium or synthetic complete (SC) medium or synthetic drop out medium (Sherman 1991). Noninduced condition consisted of

0.05% glucose, 3% glycerol and 2% lactic acid (pH5.7). For induced conditions, galactose was added to final concentration of 2% unless otherwise noted. YEP, synthetic complete or synthetic drop out media were prepared as described previously (Rose et al. 1990). Yeasts were transformed either by the one-step procedure (Chen et al. 1992) or by the high-efficiency method (Gietz et al. 1992).

Yeast strains were constructed using the methods previously described (Bahler et al. 1998, Longtine et al. 1998).

For determining the efficacy of the protein synthesis inhibitor cycloheximide using microscopic analysis, yeast strain Sc745 (Δgal4) expressing PGAL1-2GFP (two tandem copies of GFP under the GAL1 promoter) from a low copy CEN vector (pOE33) and

Gal4 (from its own promoter) also from a low copy CEN vector (pBM292) were grown to early to mid log phase in glycerol/lactic acid media and protein synthesis 54 inhibitor cycloheximide was added at 25 μg/ml final concentration (Sigma-Aldrich;

Catalog No.: C7698-5G). Thirty-minutes after cycloheximide addition, galactose was added to 2% final concentration and the cells were incubated for different lengths of time. Images were acquired immediately before adding galactose (0 hour in galactose) or 1 hour, 2 hours or 3 hours after galactose addition.

For determining the effect of protein synthesis inhibition on Gal80-2GFP cluster reassembly upon galactose withdrawal, cells were grown to early-mid log phase in

3% glycerol-2% lactic acid media and cycloheximide was added at 25 μg/ml final concentration. Thirty-minutes after cycloheximide addition, galactose was added to

2% final concentration. After one-hour incubation in galactose, cells were gently spun down at 3000 rpm and washed twice in the media without galactose and resuspended in glycerol-lactic media without galactose and with fresh cycloheximide (25 μg/ml final concentration). Images were acquired at following time points: Immediately prior to adding cycloheximide, immediately prior to adding galactose, after 1 hour incubation in galactose plus cycloheximide, after 1 hour incubation post wash, in glycerol lactic acid minus galactose plus cycloheximide and after 2 hour post wash, in glycerol-lactic acid minus galactose plus cycloheximide.

Centromeric clustering could affect the distances between two loci if they are both centromere linked (Jin et al. 2000). In this work, in a diploid strain, the distance between GAL1 and LEU2 locus was determined to see if that was significantly more 55 than the distance between the two LEU2 loci. Given that both the loci (LEU2 and

GAL1) are centromere-linked (GAL1 locus is 41 kb away from CEN2 and LEU2 is 22 kb away from CEN3), it was important that any probable effect of centromeric clustering on the distance between the two loci be reduced. It was reported that nocodazole reduces centromeric clustering (Jin et al 2000, Bystricky et al. 2005).

Therefore nocodazole (Sigma-Aldrich; Catalog No. M1404-10 MG) was used to reduce probable effects of centromeric clustering. Cells were grown to early-mid log phase in glycerol-lactic acid media and then nocodazole was added to final concentration of 15μg/ml (stock solution of 1.5 mg/ml in DMSO) and cells were incubated for three hours before image acquisition.

3.3.2 Immunoblotting: The S. cerevisiae Sc854 cells expressing Gal1-2GFP from its endogenous locus were grown to early to mid log phase in SC media (with 3% glycerol/2% lactic acid) and then cycloheximide was added to final concentration of

25 μg/ml. Galactose (2% final concentration) was added to the cells 30 minutes after cycloheximide addition and the cells were either harvested immediately or incubated for 1 hour, 2 hours, or 3 hours and then resuspended in lysis buffer (20 mM HEPES [pH 7.4], 0.5% Triton X-100, 200 mM NaCl, 0.5 mM EDTA, 2 mM dithiothreitol [DTT], 5 mM MgCl2) with protease inhibitors (1 μg/ml leupeptin,

1μg/ml pepstatin, 1 μg/ml chymostatin, 1 mM phenylmethylsulfonyl fluoride

[PMSF], 0.5 μg/ml benzamidine, and 1 μg/ml aprotinin). Acid-washed beads (0.5-

μm diameter) were added to the cells in 2-ml microcentrifuge tubes, and the cells were mechanically lysed by vortexing as previously described (Blank et al. 1997, 56 Mylin et al. 1989, Pilauri et al. 2005). An aliquot of the whole-cell extract containing

27 μg of total protein from each sample was mixed with SDS electrophoresis loading buffer and was subjected to SDS-PAGE followed by immunoblotting. The antibodies for immunoblotting were: 1:500; primary rabbit anti-GFP (Catalog No. sc8334;

Santa Cruz Biotechnology;) and 1:10,000 primary mouse anti- α-tubulin antibody

(Catalog No. T9026; Clone: DM1A; Sigma-Aldrich). Secondary antibodies (all used at a 1:10,000 dilution) Alexa-Fluor 680-conjugated goat anti-rabbit antibody

(Molecular Probes) and IR Dye 800-conjugated anti-mouse antibody (Catalog No.

610-132-121; Rockland) were used for detection by Odyssey Imaging system (LI-

COR).

3.3.3 Microscopy: Cells were grown to mid log phase (unless otherwise specified) in liquid media with appropriate carbon sources for 24 hours before imaging. Cells were immobilized on glass slide with 10% gelatin before imaging. Microscopy was performed at 23-24°C. For the experiments described in Figure 3.1, 3.2, 3.3, 3.5, 3.6 and 3.15, cells were imaged using the UltraVIEW ERS spinning disk confocal microscope (Perkin Elmer Life and Analytical Sciences, Waltham, MA) with a

100x/1.4 NA Plan-Apo objective lens (Nikon, Melville, NY). Lasers 488, 514, and

568-nm were used to excite green, yellow and red fluorescent proteins respectively.

A cooled charge-coupled device camera (ORCA-AG, Hamamatsu, Bridgewater, NJ) was used with 2x2 binning unless otherwise noted. For the experiments described in Figure 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.17 and for the estimation of number of 57 Gal4 and Gal80 molecules in their respective foci, images were collected using

100x/1.4 NA Plan-Apo objective lens on an UltraVIEW Vox CSUX1 spinning disk confocal microscope (Perkin Elmer Life and Analytical Sciences, Waltham, MA).

Lasers at 440, 488, 515, and 561 were used to excite cyan, green, yellow and red fluorescent proteins respectively. A back-thinned EMCCD camera (Hamamatsu

C91000-13, Bridgewater, NJ) was used without binning.

3.3.4 Counting the number of Gal80-2GFP clusters or Gal4-2GFP/2mYFP foci per cell: For the counting of clusters or foci as described in Figure 3.1, 3.6, and

Table 3.1 the following criteria were used to distinguish between cluster and non- cluster/smear: in general, any circular foci with clearly distinct shape and separated from the background by visual estimation was considered as cluster for Gal80 or foci for Gal4 i.e. circular-shaped feature (cluster or foci) instead of a smear or amorphous shape. To set a standard to decide on signals that are borderline low, a reference set was used that set a threshold for intensity for the peak intensity pixel

(gray value) in UltraVIEW ERS (Perkin Elmer Life and Analytical Sciences, Waltham,

MA) image analysis software and any spot with weak signal intensity but circular in shape and small size is not considered as cluster if the intensity of the peak intensity pixel (single “Z” plane) is less than 230.

3.3.5 Standard for true colocalization: The standard for true colocalization was established using the well-characterized system used previously in the J Hopper lab

58 to visualize Gal4, Gal80 and Gal3 in the nucleus at the GAL1 promoter. The P - GAL1

GSTx 8 containing array of 8 tandem repeats of GST gene under the GAL1 promoter was inserted approximately 9.7 kb away from the LacOx64 array that contains 64 tandem repeats of the Lac operator sequences, (Jiang et al. 2008). This leads to the formation of Gal80-2GFP dot, which is typically more compact and less varied compared to Gal80-2GFP clusters, on P -GSTx 8 array. This Gal80-2GFP dot GAL1 colocalizes with the LacOx64 bound LacI-mCherry dot as described previously

(Jiang et al. 2009, Egriboz et al. 2013). In this work, the distances between the midpoints of the promoter regions/UASGAL sites of all the loci studied and the midpoint of the inserted LacO array ranges from 7-10 kb, so it does reflect a similar spatial relationship and hence the standard was considered applicable for determining true colocalizaton. The yeast strain Sc913 containing both the P - GAL1

GSTx 8 tandem repeat array as well as expressing 2GFP tagged (at 3’ end) Gal80 and the LacI-mCherry was grown to mid log phase before image acquisition. For image acquisition, 11 Z stacks were taken with the Z-spacing of 0.4μm. Over 300 cells were analyzed to determine the relationship between the peak intensity pixels of the

Gal80-2GFP dot and the LacI-mCherry dot. Based on this analysis, it was observed that there are two features regarding the spatial relationship between the peak- intensity pixels of the two fluorescent dots that are conserved in > 99% of the cells.

The two features are: the peak intensity pixels of the two fluorescent dots are on the same Z-plane and the two peak intensity pixels either coincide or are just next to each other as shown in Fig 4F. These two features were used to formulate the

59 standard for colocalization as follows: A. The peak intensity pixels for both the fluorescent feature (cluster or dot) should be on the same Z-plane. B. The peak intensity pixels of the two fluorescent feature should either coincide or be next to each other as shown and described in Figure 3.4F. Moreover, to rule out any bias in the analysis, the image acquisition and analysis were both done blind with no knowledge of what particular strain is being imaged or analyzed until the end of analysis of all the data.

3.3.6 Estimation of the number of molecules in the Gal80 cluster and in the

Gal4 foci using fluorescent intensity measurements: Estimation of the number of molecules in Gal80 clusters and Gal4 foci were determined using a previously developed standard described in Coffman et al (2011). Cells were grown to mid log phase before imaging. Total 12-Z sections were taken with the Z-spacing of 0.4μm with no binning. Correction for background fluorescence, DC offset and Dark current was carried out as per the manuals in Volocity user guide

(cellularimaging.perkinelmer.com/pdfs/manuals/ Volictyuserguide.pdf). Briefly, a dark reference image was acquired from an area of the sample that contains no cells or debris in the image field under the setting that was used to acquire images of the cells. This dark reference image was subtracted from all the images of that particular sample before calculation of fluorescence intensity. Fluorescence intensity was calculated using imageJ (http://imagej.nih.gov/ij/) software.

Integrated fluorescence intensity (minus background or BG) was obtained as described previously (Lawrimore et al. 2011): Briefly, a 5x5 pixel region was 60 centered on the fluorescent feature (cluster or foci) to obtain integrated fluorescence, whereas a 7x7 region centered on the 5x5 region was used to obtain surrounding BG intensity using ImageJ (http://imagej.nih.gov/ij/). For each feature, sum intensity of three consecutive Z-sections was obtained where the Z-section with the maximum intensity was in the middle. Measured values were calculated as: integrated fluorescence intensity (minus BG) = integrated counts for 5 x 5 region –

(integrated counts for 7x7 region-integrated counts for the 5x5 region) x pixel area of 5x5 region/(pixel area of 7x7 region-pixel area of 5x5 region). Thus background measurements were obtained from the value of 7x7 pixel region minus the value of

5x5 region and scaled to the same area as the specimen.

The above method was used for the determination of the fluorescent intensity for all quantitation. Additionally, for the estimation of the number of molecules in Gal80 clusters and Gal4 foci, images of the standard Cse4-mYFP (JW2687) (Coffman et al.

2011) were obtained and fluorescence intensity of Cse4-mYFP in anaphase cluster of 100 cells were quantified and the average was used as reference standard as 122 molecules as described in Coffman et al (2011). For Gal80-mYFP and Gal4-2mYFP

(final values for Gal4-2mYFP was divided by 2 as there are 2 mYFP molecules fused to one Gal4 molecule), fluorescence intensity was determined for 50 cluster or foci each. All the images were acquired under the same setting and side-by-side. The standard and the sample could not be mixed, because the strain expressing standard

Cse4-mYFP (JW2687) (Coffman et al. 2011) had to be grown in 2% glucose as it grew very poorly in glycerol-lactic acid media and the signals from Cse4-mYFP 61 clusters were weak. On the other hand both the Gal4 (Sc1160) and Gal80 (Sc841) had to be grown in the absence of glucose as their expression would have been severely repressed or affected in glucose. To avoid any additional variability, images were acquired side by side with minimal time gap between the standard and the test samples. However, for comparison of fluorescence intensity of Gal4-2mYFP, in the presence (Sc1160) and absence (Sc1161) of Gal80, the cells were mixed and were imaged on the same slide. The strains were distinguished by expressing different fluorescent protein markers in the two strains (LacI-mCherry and PP7-2CFP).

3.3.7 Measuring distances between the two fluorescent dots of Tet repressor

GFP (TetR-GFP) and LacI-mCherry: Distances between the TetR-GFP and the LacI- mCherry dots were determined by measuring the 3-D (three-dimensional Euclidean distance) distances between the centroids of the two dots. The images were acquired with 0.2μm Z-spacing and in total 21 Z slices were taken per image with no binning. The co-ordinates of the centroid of each spot were determined using object finder under the measurement tool in Volocity software for image analysis (Perkin

Elmer Life and Analytical Sciences, Waltham, MA). Distances in pixels were converted to μm using 14.4 pixel/μm conversion factor. To compare between different distributions and obtaining the p-value, the Kolmogorov-Smirnov test was carried out.

62

3.4 RESULTS:

3.4.1 Gal80-2GFP predominantly makes 1, 2, or 3 intranuclear clusters per cell:

As mentioned in Chapter 2, Gal80 tagged in frame at its 3’ end with 2GFP/2mYFP shows intranuclear clusters in glycerol-lactic acid media and these clusters were found to dissipate upon addition of galactose. Initial effort of this work was directed towards characterization of the basic features of these clusters using much larger sample size than previously done. It was observed that Gal80-2GFP predominantly makes 1, 2 or 3 (more than 85% cells - N = 300) circular shaped clusters per cell as shown (Figure 3.1A) but infrequently 4 or more clusters were also observed (data not shown). Typically, the solitary cluster in the cells with only one cluster appeared to be bigger than the clusters in the cells with more than one cluster. It could be speculated that a single cluster may be formed through association of two or more smaller clusters. Gal80-2mYFP as well as Gal80-mYFP exhibited similar shape and number distribution as Gal80-2GFP (data not shown). This suggests that the fluorescent protein tag is not significantly affecting the behavior of the Gal80 cluster as GFP has a much higher affinity for dimerization than mYFP (monomeric YFP)

(dissociation constant for GFP is 0.11 mM whereas for monomeric YFP it is 74 mM – 63 Zaccharias et al. 2002), additionally mYFP and GFP differ in their quantum yield as well. Furthermore, it was observed that the Gal80 clusters showed a lot of variability with respect to number, size, shape and intensity and also seemed to be quite dynamic as shown in Figure 3.1B.

Fluorescent quantitation method as described by Coffman et al. (2011) was used to obtain an estimate for the number of Gal80 molecules in these clusters. This showed that the number of Gal80 molecules in these cluster ranges from 20-100 with average being 60 (n=50) indicating that Gal80 clusters are large molecular assemblies.

3.4.2 Gal80-2GFP clusters are observed in raffinose but not in glucose:

The effects of the repressing carbon source glucose and the non- inducing/nonrepressive carbon source raffinose were tested on Gal80-2GFP clusters. When grown in glucose, Gal80 clusters were rarely visible and when observed, were extremely weak in intensity (Figure 3.2A). On the other hand, in raffinose, which is similar to glycerol-lactic acid in being both non-inducing and nonrepressive for the transcription of the GAL genes, Gal80 made clusters similar to those observed in glycerol-lactic acid (Figure 3.2B). The absence of any clear visible cluster of Gal80 in glucose is somewhat surprising because Gal80 expression was reported to be similar in glycerol and glucose based on its mRNA level (Shimada and

Fukasawa 1985). If the expression of Gal80 is indeed similar in both these carbon sources, then there must be some other component/s that is required for Gal80 64 cluster formation, which is affected differently by these two carbon sources. One such candidate is Gal4 itself because Gal4 expression is tightly repressed in glucose

(Griggs and Johnston 1991). This possibly implicates a role for Gal4 in the formation of Gal80 cluster.

3.4.3 Preexisting Gal80 can reassemble into cluster upon galactose depletion:

From the previous work, it is known that upon removal of galactose, Gal80 clusters reappear in glycerol-lactic acid media (Egriboz et al. 2013). In that work reassembly of dissipated Gal80 clusters occurred even under the condition where the protein synthesis inhibitor cycloheximide was used in an attempt to block Gal80 protein synthesis. However, in those previous experiments, the cycloheximide was not actually checked for being effective in blocking protein synthesis. Therefore, experiments were carried out to determine if a newer batch of cycloheximide used at 25μg/ml concentration could block protein synthesis in galactose media for two hours (the duration of the experiment) after addition. The tight repression in the absence of galactose and the rapid and robust induction in the presence of galactose of the GAL1 promoter was utilized to test the efficacy of cycloheximide. In these experiments, cycloheximide was added 30 minutes prior to the addition of galactose and the cells were incubated for different lengths of time (0 hour, 1 hour, 2 hours and 3 hours). Immunoblotting and microscopic analysis were employed to check the efficacy of cycloheximide. With the immunoblotting, it was observed that the 65 expression of the Gal1-2GFP was severely repressed even 3 hours after addition of galactose in the presence of cycloheximide (Figure 3.3A). Also, with the microscopic analysis, the expression of the PGAL1-2GFP was found to be severely repressed in the presence of cycloheximide (Figure 3.3B) suggesting that cycloheximide at 25μg/ml concentration could block protein synthesis for at least 3 hours after addition. Once the efficacy of the cycloheximide was established, it was asked if the preexisting

Gal80 molecules could reassemble to form clusters upon removal of galactose in the absence of synthesis of new Gal80-2GFP molecules. It was observed that the Gal80 clusters dissipate upon addition of galactose but complete disappearance of the clusters requires incubation in galactose for 30-45 minutes. Therefore, to ensure complete dissipation of the Gal80 clusters, the cells were incubated in galactose for one hour before returning them back to glycerol lactic acid media (Figure 3.3C) in the presence of cycloheximide. Upon removal of galactose and in the presence of cycloheximide, Gal80 clusters did not reappear within one hour after galactose removal, but rather reappeared after two hours (Figure 3.3D and 3.3E respectively).

This result indicates that the pre-existing pool of Gal80 molecules can reassemble to form clusters upon galactose removal.

66 3.4.4 Gal80 clusters colocalize with the UASGAL1-10-7 locus:

One important question regarding Gal80 cluster formation was: “what is the basis for formation and/or maintenance of such clusters?” Because Gal80 binds to Gal4 but not to DNA (Lue et al. 1987, Platt and Reece 1998) and Gal4 binds specifically to the UASGAL sites (Bajwa et al. 1988, Bram and Kornberg 1985, Bram et al. 1986,

Giniger et al. 1985, Lohr and Hopper 1985), a prime candidate for nucleating Gal80 clusters is Gal4 associated with the UASGAL sites. This hypothesis is encouraged by the fact that similar clustering is observed with proteins that are tethered to some

DNA element in the genome e.g. Sir3, Sir4, and Rap1 form the so-called perinuclear foci associated with telomeric mediated by Rap1 binding to telomere sites (Klein et al. 1992; Palladino et al. 1993; Gotta et al. 1996;Laroche et al. 1998). Gal80 does not directly bind to DNA but it interacts with Gal4 ((Lue et al.

1987, Platt and Reece 1998). So, the general approach was to first determine the frequency of colocalization of Gal80-2GFP cluster with the UASGAL loci. Any particular UASGAL locus was monitored through the visible dot formed by binding of

LacI-mCherry to the LacOx64 array (an array of tandem repeat of 64 Lac operator sequence) inserted downstream of the UASGAL –associated GAL gene as shown in the diagram in Figure 3.4A. Given that GAL1, GAL7, GAL10 (GAL1-10-7) on

Chromosome II has the largest concentration of UASGAL sites (six) in close proximity

(Figure 3.4B), it was checked if Gal80 cluster colocalizes with this region. To ascertain true colocalization and avoid false positive error, a well-characterized system developed in the Jim Hoper lab to visualize Gal4 and Gal80 in the nucleus on 67 the GAL1 promoter (Jiang et al. 2009) was used to formulate a standard for true colocalization as shown in Figure 3.4C and 3.4D. The details are provided in the materials and methods section of this chapter. To avoid any bias in calculating the frequency of localization, the image acquisition and analysis were carried out blind.

The LEU2 locus with no known UASGAL site was used to determine the frequency of random colocalization. Consistent with the hypothesis, it was observed that Gal80-

2GFP strongly colocalizes with the GAL1-10-7 locus (78% - strain Sc1020), which is significantly higher (p<0.001) than colocalization with the LEU2 locus (44% - strain

Sc1082) (Figure 3.5A). Importantly, the UASGAL sites associated with the GAL1-10-7 locus are required for such high level of colocalization as deletion of all six UASGAL sites (strain Sc1031) dropped the colocalization from 78% to 29%. Unexpectedly, deletion of only the four UASGAL sites of the divergent region (DVR) within the GAL1-

10-7 locus (Sc1021) or the two UASGAL sites in GAL7 promoter (Sc1032) separately did not decrease colocalization. So, it appears that at least the two UASGAL sites of the

GAL7 promoter region or the four UASGAL sites of the GAL1-10 DVR are required for the observed high level of colocalization of the GAL1-10-7 locus with the Gal80-

2GFP cluster.

As a follow-up to these results, the role of the UASGAL sites in colocalization of a locus with Gal80-2GFP clusters was further investigated. The four UASGAL sites of the DVR spanning 140 bp were inserted into the ectopic LEU2 locus which without the DVR showed low level of association with Gal80-2GFP as shown in both Figure 3.5A and

3.5B (left most bar). The LEU2 locus with the inserted DVR (Sc1041) showed 68 significantly higher (63%, p = 0.0067 compared to 44% without the DVR) colocalization with Gal80-2GFP cluster (Figure 3.5B second bar from left). However the colocalization frequency was not as high as with the native GAL1-10-7 locus at its normal chromosomal location. One possible explanation is that insertion of the four UASGAL sites in the LEU2 locus could lead to competition between two sets of four UASGAL sites for a fixed pool of Gal80-2GFP molecules. To test this idea, the four

UASGAL sites from the GAL1-10 promoter (Sc1050) were deleted before checking the colocalization frequency of the LEU2 locus with the DVR inserted. Indeed, it was observed that there was a significant increase in the colocalization frequency of the

LEU2 locus from 63% to 84% (p = 0.0005) (Figure 3.5B-third bar from left). To evaluate this result further, the DVR was inserted into the GAL3 locus which has only one native UASGAL site (Sc1044) and colocalizes with Gal80-2GFP cluster with a low frequency (43%-fourth bar from the left) similar to the 44% frequency for the

LEU2 locus (see above) and found that the insertion of the DVR into the GAL3

(Sc1084) locus resulted in a significant increase in the colocalization frequency from

43%-85%, (p <0.0001, Figure 3.5B). Taken together, these results suggest that insertion of the four UASGAL sites of the GAL1-10 DVR into the LEU2 or the GAL3 loci is sufficient to increase the frequency of colocalization with Gal80-2GFP clusters.

And these results strongly suggest that there may be competition for Gal80-2GFP clusters at different UASGAL sites within the genome. Furthermore, it appears that the presence of just one UASGAL site (like in the GAL3 locus) is not sufficient for the high frequency of colocalization of the locus with a Gal80 cluster.

69 3.4.5 Deletion of UASGAL sites in GAL1-10-7 does not affect the number distribution of Gal80-2GFP cluster within the yeast population:

Results presented in the last experiment strongly implicated UASGAL sites as nucleation sites for Gal80-2GFP cluster formation and/or maintenance. Accordingly, it was expected that deletion of the six UASGAL sites associated with the GAL1-10-7 locus would impair Gal80-2GFP cluster formation or maintenance. Surprisingly, however, deletion of all six UASGAL sites showed no effect on the number of intranuclear Gal80-2GFP clusters per cell (Figure 3.6) although the association of the GAL1-10-7 locus with a Gal80-2GFP cluster dropped significantly (29%) as shown in Figure 3.5A. Thus the relationship between the number of UASGAL sites (as in the GAL1-10-7) and the occurrence of GAL80-2GFP clusters is more complex than anticipated.

3.4.6 Gal80-2mYFP cluster formation is dependent on Gal4:

As mentioned previously, Gal80 does not bind to UASGAL DNA sites, but rather to

Gal4, therefore, it was important to determine whether occurrence of Gal80-2GFP cluster is dependent on Gal4. Gal80 expression itself is Gal4 regulated and deletion of Gal4 can affect Gal80 expression level and thereby can indirectly affect clustering.

So to evaluate the requirement of Gal4 on the occurrence of Gal80 clusters, Gal80 70 was needed to be expressed at a level similar to its endogenous level in glycerol lactic acid media but from a Gal4-independent promoter. Gal80-2mYFP expressed from STE50 promoter showed clusters very similar to those formed when Gal80-

2mYFP was expressed from its own promoter. When Gal80 was expressed from this

STE50 promoter (pSG45), it was observed that Gal80-2mYFP made clusters in the presence of Gal4 but not in the absence of Gal4 (Figure 3.7) indicating Gal4 or a

Gal4-dependent gene product is required for Gal80 clustering.

3.4.7 What is the nature of requirement of Gal4 for Gal80 cluster formation?

The observations that Gal80-2GFP clusters associate with the UASGAL1-10-7 with very high frequency and that clustering is dependent on Gal4 suggest that Gal4 might be providing a bridge between the underlying DNA and the Gal80-2mYFP by binding to both the DNA and Gal80 simultaneously (Lue et al. 1987, Platt and Reece 1998,

Bajwa et al. 1988, Bram and Kornberg 1985, Bram et al. 1986, Giniger et al. 1985,

Lohr and Hopper 1985). So one reasonable hypothesis to test was whether Gal4 could be replaced by insertion of DBD (GAL4 aa 1-147) to Gal80. To address this,

DBD-Gal80 was expressed under GAL4 promoter from a low copy CEN vector

(pEB1) in a gal4 deletion strain where the endogenous GAL80 is tagged in-frame at its 3’ end with 2mYFP (sc1068). Gal80 clusters were visible in the presence of this

DBD-Gal80 even in the absence of Gal4 but the clusters looked somewhat weak in intensity (Figure 3.8A). The intensity of the clusters increased when PGAL4 DBD-

71 Gal80 was replaced with PGAL4 DBD-Gal80-2mYFP (pEB2) (Figure 3.8B) where both the endogenous Gal80 and the PGAL4 DBD-Gal80 were tagged with 2mYFP.

3.4.8 PGAL4 DBD-Gal80 can make intranuclear cluster in the absence of both

Gal4 and Gal80:

Next it was asked, if PGAL4 DBD-Gal80-2mYFP can yield clusters on its own in the absence of both Gal4 and Gal80. To address this, PGAL4 DBD-Gal80-2mYFP was expressed in a gal4 gal80 deletion strain (Sc1153). This construct did indeed yield typical intranuclear clusters (Figure 3.9A). So clearly one role of Gal4 for Gal80 cluster formation is to provide a link to UASGAL sites through its DBD.

3.4.9 Is DNA binding required for formation of PGal4 DBD-Gal80-2mYFP clusters?

Given that PGAL4 DBD-Gal80-2mYFP can make intranuclear clusters, one important question that remained was whether DNA binding is necessary for such cluster formation. The DBD of Gal4 consists of aa 1-147, but the DNA binding determinants reside within aa 1-65 (Marmorstein et al. 1992). A well-characterized mutant of

Gal4 DBD, i.e. L32P that was previously shown to be impaired in DNA binding

(Johnston and Dover 1987) was used to address this question. It is also important to 72 note that in a separate study Silver et al (Silver et al. 1987) found that the Gal4 nuclear localization signal (NLS) overlaps with the DNA binding domain and one particular amino acid C38 (Cystein38) through different mutation affects both the

DNA binding and the nuclear localization of Gal4 (Johnston and Dover 1987, Silver et al. 1988). Interestingly when DBD L32P mutant variant of PGAL4 DBD-Gal80-

2mYFP (pSG47) was used, it was observed that its nuclear localization is severely impaired (Fig 3.9B). This is seemingly a novel observation. There has been no known evidence in the literature of its effect on the nuclear localization of Gal4 but its pattern of distribution in the cell bears great resemblance with the distribution pattern under immunofluorescence assay of some of the mutants that Silver et al.

(1988) identified in the same region.

Since there is no known mutant that affects DNA binding without affecting nuclear localization, the next question was whether Gal80-2mYFP can yield cluster in the absence of Gal4 if Gal4 DBD in PGAL4-DBD-Gal80-2mYFP is replaced by SV40 NLS

(simian virus nuclear localization signal). It was observed that PGAL4 NLS-Gal80-

2mYFP (pSG48) did not show any cluster (Figure 3.9C) suggesting but not proving that the DNA binding function might well be required for formation of intranuclear cluster. This result does indicate however that NLS cannot replace DBD for the formation of Gal80 cluster.

73 3.4.10 Does occurrence of Gal80-2GFP clusters depend on Gal80 self- association?

The next question addressed was whether self-association is required for formation of Gal80 clusters. Gal80 self-associates and binds to Gal4 as a dimer (Melcher and Xu

2001, Kumar et al. 2008). Thus it was of interest to determine if Gal80 self- association is required for cluster occurrence. In an attempt to address this, a previously characterized mutant of Gal80, Gal80N230R was used that had been shown to disrupt self-association (Kumar et al 2008). PSTE50 Gal80-2mYFP N230R

(pSG46) failed to show any cluster (Figure 3.10B). However, its interpretation is difficult since Gal80N230R is also severely impaired in its interaction with Gal4

(Kumar et al. 2008) and hence it is not clear if impairment of self-association or binding to Gal4 or both were the reasons for no cluster formation. In fact, the issue of whether Gal80 self-association is involved in cluster formation remains a challenge because all the current mutants that are known of Gal80 that impair self- association also impair association with Gal4 (Pilauri et al. 2005, Goswami and Jin unpublished data).

3.4.11 Gal4-2GFP and Gal4-2mYFP make intranuclear foci both in the absence and presence of galactose:

Clustering of PGal4 DBD-Gal80-2mYFP was initially thought to be due to self- association of Gal80 molecules. Gal4 is a very strong dimer not known to form higher order oligomers (Carey et al. 1989) and thus it was not anticipated that Gal4- 74 2GFP would yield clusters like PGAL4 DBD-Gal80-2mYFP or Gal80-2mYFP.

Surprisingly, Gal4-2GFP when expressed under its own promoter and expressed from a low copy CEN plasmid (pFJ110N) showed bright intranuclear foci (Figure

3.11A) which unlike Gal80-2GFP clusters did not dissipate even after one-hour incubation in galactose (Figure 3.11B). It should be pointed out that Gal4-

2GFP/2mYFP assemblies were referred to as foci just to make the distinction with

Gal80 clusters, they looked similar in many cells, but in general Gal80-2GFP clusters are bigger in size than Gal4-2GFP foci and also Gal4-2GFP foci showed much less variability in size and intensity compared to Gal80 clusters (data not shown).

3.4.12 Formation of Gal4-2GFP/2mYFP foci is not dependent on Gal80

When tagged in frame at its 3’ end with either 2GFP or2mYFP and expressed from its endogenous locus, Gal4 makes intranuclear foci both in the presence and absence of Gal80. Gal4-2mYFP seems to make more foci than Gal4-2GFP (Table 3.1) but in most of the cells there was one focus with much higher signal intensity compared to other foci if other foci were indeed present. The difference in the number of Gal4-

2GFP and Gal4-2mYFP foci could be due the difference in quantum yield between the two fluorescent proteins GFP and mYFP.

Photon counting using the method mentioned previously (Coffman et al. 2011) indicated that there are about 45 molecules of Gal4 on average in the brightest focus although the number of molecules in each assembly ranged from 20 to 60. 75

3.4.13 Gal4 makes intranuclear foci independent of Gal80 but are they of equal intensities in the presence and absence of Gal80?

This question was addressed by measuring the fluorescence intensities of Gal4-

2mYFP foci in Gal80 WT and Δgal80 strains. The result as shown in Figure 3.12 bar chart indicates that there is no statistically significant difference in the intensities between Gal4-2mYFPs in WT Gal80 cells and in Δgal80 cells.

3.4.14 The brightest Gal4-2GFP focus colocalizes with the GAL1-10-7 locus:

As mentioned before, fluorescent-tagged Gal4 typically showed one focus brighter than the rest. It was hypothesized that this bright focus is associating with the GAL1-

10-7 loci. It was observed that in 100 cells with bright Gal4-2GFP foci (Sc1163) and a visible LacI-mCherry dot (marking GAL1-10-7 locus), the colocalization was 100% as shown in Figure 3.13A. If Gal4 binding to UASGAL sites is mediating this association, one would expect that deletion of the UASGAL sites in this locus would significantly reduce the association. As expected, a significant drop in the colocalization frequency was observed in ΔGAL1-10-7 where all the six UASGAL sites were deleted (Sc 1205). A highly significant change in the focus intensity was also observed (Figure 3.13B) with very few cells showing any kind of foci (28/100) and amongst these faint foci only 3 showed association with the LacI-mCherry dot (only

76 3%) reinforcing the idea that the bright Gal4 focus is associating with the UASGAL sites at the GAL1-10-7 locus.

3.4.15 Gal80 clusters and Gal4 foci can be simultaneously observed in the same cell:

The fact that Gal80-2mYFP/2GFP clusters and Gal4-2mYFP/2GFP foci can be observed under the microscope posed two related questions. Firstly, can one observe both the Gal80 and Gal4 foci in the same cell? One would expect so. And, if yes, the next question is whether Gal80 cluster and Gal4 foci reflect basically the same phenomenon of the UASGAL-bound Gal4 interacting with Gal80. If yes, then one would expect to observe that the cells contain equal number of Gal4 foci and Gal80 clusters and that the assemblies of the two proteins always colocalize with each other.

To address whether both fluorescent protein tagged Gal4 and Gal80 assemblies can be discerned in the same cell, endogenous Gal80 was tagged with tdTomato in frame at its 3’ end in a strain with the genomic Gal4 tagged at its 3’end with 2GFP

(Sc1165). Both fluorescent-protein tagged Gal4 and Gal80 assemblies were visible in glycerol-lactic media (Figure 3.14A) and as expected Gal80-tdTomato but not 77 Gal4-2GFP showed dissipation in galactose (Figure 3.14B). Interestingly, when all the six UASGAL sites in the GAL1-10-7 were deleted in the same strain (Sc1205), it showed a strong effect on Gal4 foci, but much less of an effect on Gal80 clusters as shown in Figure 3.14C. The behavior of the two proteins in both the WT and the

∆GAL1-10-7 background were recorded and the results are presented in Table 3.2.

In the WT background, a high percentage (59%) of cells showed complete colocalization of Gal80 clusters with Gal4 foci. In contrast, in the background of

ΔGAL1-10-7, the numbers were very different where only 19% of the cells showed complete colocalization as shown in Table 3.2. This suggests that although one can visualize both proteins as distinct cluster or foci, the two assemblies are not always associating in equal numbers. This aspect will be elaborated in the discussion section.

3.4.16 Do the large molecular assemblies of Gal80 constitute bridges between distant UASGAL sites?

Several previous studies showed that Gal80 can self-associate independently of Gal4 both in-vitro and in-vivo to form higher order structures (Melcher and Xu 2001,

Pilauri et al. 2005, Lavy et al. 2012, Egriboz et al. 2013). The photon counting of

Gal80-mYFP clusters indicates the presence of a very large number of Gal80 molecules in these assemblies. This raises the possibility that these molecular assemblies of Gal80 can potentially act as a bridge between different UASGAL sites 78 mediated by two interactions, one with Gal4 and the other with another Gal80 molecule. The idea is that a dimer of Gal80 complexed with Gal4 might associate with a non-Gal4-bound Gal80. If there is such bridging between different UASGAL sites then they are expected to be closer to each other than to any random locus.

Moreover, given that Gal80 associates most strongly with the GAL1-10-7 locus, it is reasonable to expect that in a diploid strain, a GAL1-10-7 locus would be located significantly closer to another GAL1-10-7 locus compared to a random locus with no known UASGAL site. To test this idea, the following experiment was designed, the basic scheme of which is as depicted in the diagram in Figure 3.15. First the two loci of interest were tagged in two haploids of opposite mating type. One was tagged with a Tet operator array of tandem repeats of 512 Tet operator sequences,

(TetOx512) and the other one was tagged with a Lac operator array (LacOx64).

Insertion of tags ensured that upon expression of Tet Repressor GFP (TR-GFP – pSG50) it will bind to the Tet operator array and form a visible fluorescent dot, similarly LacI-mCherry (pSG51) will bind to the LacOx64 array and make a visible dot marking the tagged locus. Upon mating the two differently tagged strains of opposite mating type, a diploid would be obtained where one can visualize both the loci under investigation. With this system, the frequencies of colocalization were determined between the following loci: two LEU2 (in the diploid upon mating of

Sc1124 x Sc1134) loci (used as a random non UASGAL-containing locus), one LEU2 locus with a GAL1 locus (in the diploid upon mating of Sc1122 x Sc1134) and one

GAL1 locus with another GAL1 locus (in the diploid upon mating of Sc1123 x

Sc1134). Initial results indicated that there was no significant statistical difference 79 in the frequency of colocalization between LEU2-GAL1 vs. GAL1-GAL1 (frequency of colocalization between the two GAL1 loci was 31% and between GAL1 and LEU2 loci was 26%; p = 0.4366; n=100). The LEU2 and the GAL1 loci share one common feature i.e. they are both centromere linked (GAL1-10 about 41 kb away from CEN2 and Leu2 locus is about 22 kb away from CEN3), thus may be part of the centromeric cluster (Jin et al. 2000) and thus show higher than random level of colocalization with each other. Additionally, colocalization analysis would only give a binary result (colocalized or “not” colocalized) and place everything that is not colocalized under one category. However, the two loci could come quite close to each other through bridging but may not actually colocalize. Therefore, to have a more accurate estimate of the distance relationship between the two loci, determination of the 3-D distances between the centroids of the two fluorescent spots seemed to be more the more appropriate approach. Nocodazole treatment was shown to reduce centromere clustering (Jin et al. 2000), possibly by disrupting the short microtubules that attach the centromeres to the spindle pole body (SPB) in the interphase (Jin et al. 2000, Bystricky et al. 2004) Therefore, nocodazole was used to reduce the possible effect of centromere clustering on the measured distances between the loci of interest. The 3-D distances between the following centroids were measured: between LEU2-LEU2, LEU2-GAL1, and GAL1-GAL1 with or without nocodazole (with DMSO). The two-sample Kolmogorov-Smirnov test (KS test) was used to compare between the two distance distributions and determine the p value for significance testing. In the absence of nocodazole, at 95% confidence level the two GAL1 loci were found to be significantly closer compared to GAL1 to 80 LEU2 (p = 0.039; Table 3.5). Interestingly, the two LEU2 loci were also significantly closer than LEU2 and GAL1 loci (p = 0.016) and there was no significant difference in distances between LEU2-LEU2 and GAL1-GAL1 (Table 3.5). With nocodazole treatment, a very different result was obtained. Firstly, the average distances between the two LEU2 loci as well as between the two GAL1 loci increased (Table

3.6) whereas for LEU2-GAL1 it decreased. Secondly, as a consequence of that, there was no significant difference (at 95% confidence level) in distances between a LEU2 and a GAL1 locus compared to two GAL1 loci but now the two GAL1 loci were closer than the two LEU2 loci (Table 3.7). These results suggested that a GAL1 locus is not significantly closer to another GAL1 locus compared to a random (LEU2) non-

UASGAL -containing locus. Additionally, it also suggested that the centromeric clustering might be affecting the distance between the two LEU2 loci since nocodazole treatment had the most pronounced effect on the LEU2-LEU2 distance distribution. It should be pointed out that although both GAL1 and LEU2 are centromere linked, LEU2 is much closer to the CEN3 (22 kb) than GAL1 to CEN2 (41 kb). Taken together, these results did not provide any definitive evidence that there is bridging between two UASAGAL containing loci.

3.4.17 Is Gal80 clustering correlated with tighter repression?

Several lines of evidences suggest that Gal80 self-association may be important for its inhibition of Gal4 activity (Melcher and Xu 2001, Pilauri et al 2005, Kumar et al.

2008, Egriboz et al. 2013). In this work, Gal80-2GFP/2mYFP clusters were found to associate strongly with the GAL1-10-7 locus. It is also well known that the 81 transcriptions of GAL1, 10, and 7 are very tightly repressed by Gal80 in the absence of galactose. A possible functional significance of this is that due to their containing such large number of Gal80 molecules and associating with the GAL1-10-7 locus they might confer tighter repression akin to what is suggested for the E. coli gal repressor (repressosome hypothesis) (Aki and Adhya 1997, Lewis and Adhya 2002).

Finding a correlation between the repression of transcription of a GAL locus and its association with the cluster on one hand and activation of transcription from the locus when it is not associated with the cluster on the other would be consistent with such an idea. As previously noted, complete dissipation of Gal80-2GFP or

Gal80-2mYFP cluster in 2% galactose takes from 30-45 minutes with some cells showing dissipation pattern earlier than others. However, it is not known if such dissipation is associated with the activation of transcription of GAL genes or if they are two completely independent events. It was hypothesized that at a lower galactose concentration (much less than 2%), cluster would take longer time to completely dissipate. Therefore at a lower galactose concentration, at early time period after galactose addition, more cells would retain clusters than they would in the presence of 2% galactose. This persistence of cluster for additional time would leave more cells with cluster after galactose addition and yet would still activate transcription from the GAL genes in many cells, which can be monitored at the same time. This would allow one to ask if the persistence of association of a cluster associated with a locus is correlated with transcriptional repression. To accomplish this, one needed to monitor the transcriptional state of the gene simultaneously with Gal80-2mYFP cluster and the locus (through LacI-mCherry). For this, a system 82 originally developed in the Singer Lab (Larson et al. 2011) to detect newly synthesized transcript was utilized. This system utilizes an RNA-binding protein and its recognition site tagged at the transcript being monitored. This is depicted in the diagram in Figure 3.16. PGAL1-GST with 24 PP7 binding sites (PP7BS x 24) fused at 3’ end (pOE232) was inserted at the LEU2 locus in a strain, which already contained

LacOx64 inserted at the LEU2 locus and Gal80 tagged at its 3’ end with 2mYFP.

Once the PP7BS x 24 region is transcribed the PP7 part of the transcript will form stem-loop structure which will be recognized by the PP7 RNA-binding protein (PP7-

CP) and the protein will bind to the RNA transcript. Use of fluorescent protein- tagged version of PP7 (pSG49: PP7-2CFP) would allow visualization of the transcript in real time as well as the LEU2 (LacI-mCherry dot) locus and the GAL80-2mYFP cluster. As shown in Figure 3.17A, in glycerol-lactic media, the LacI-mCherry dot colocalizes with the Gal80-2mYFP cluster and there is no visible PP7-2CFP dot suggesting repressed state of inserted PGAL1-GST. The cells were induced with galactose to final concentration of 0.05% for the reasons mentioned earlier and incubated for 20 minutes before image acquisition. Mainly three types of cells were observed. One group showed no transcript dot with Gal80-2mYFP colocalizing with

LacI-mCherry (Figure 3.17B), another group showed neither Gal80-2mYFP colocalization with LacI-mCherry nor any transcript dot (Figure 3.17C white arrow) and the third group showed PP7-2CFP transcript dot next to the LacI-mCherry dot which was not associated with Gal80-2mYFP (Figure 3.17C colored arrow). It is interesting to note that for the latter, two distinct Gal80-2mYFP clusters flank the

LacI-mCherry dot but neither of them is actually colocalizing with the LacI-mCherry 83 dot. It is to be noted that the 3’end tagging of PP7 BS results in more transient association of the visible transcript dot with the locus than with the 5’end tagging with PP7BS. Most of the time, the transcript dot was observed next to the locus and not colocalizing with the LacI-mCherry dot marking the locus. The results from randomly selected 103 cells are shown in Table 3.4. It was observed that the cells either showed A. Transcript dot with no Gal80-2mYFP associating with the locus

(47%), or B. No transcript and the locus is colocalizing with Gal80-2mYFP cluster

(32.07%), and C. Around 20% cells showed neither Gal80-2mYFP cluster colocalizing with the locus nor any transcript. Strikingly, less than 2% (out of 103 cells), showed visible transcript even when a cluster is associated with the locus.

Although these results are not conclusive, they are encouraging to the extent that they indicate that the presence of Gal80 cluster on the GAL gene locus might be correlated with transcriptional repression. Transcript dots are only visible in cells with no visible clusters indicating dissipation event has already taken place. It should be emphasized that this is a preliminary result and more experiments with larger sample sizes are required before one can make any definitive conclusion on this aspect.

84 3.5 DISCUSSION:

In this section, the high spatial resolution of spinning disk confocal microscopy was used extensively to investigate the behavior of the newly discovered intranuclear clusters of the transcriptional inhibitor Gal80. It was observed that Gal80 when tagged with either 2GFP or 2mYFP made intranuclear clusters with predominantly

1, 2, or 3 distinct clusters per nucleus and the clusters exhibited a lot of variability in terms of number, size, shape, and intensity. Moreover, as the figure 3.1B shows, they also appear to be quite dynamic. This variability and dynamic nature make it very difficult to rigorously define what is a Gal80 cluster. Therefore, it was determined largely empirically as described in the results section. Based on the fluorescence- quantitation-based estimation, the number of Gal80 molecules in these clusters was found to vary from 20 to 100 with the average being 60. It is to be noted that Gal80-

2mYFP/2GFP tagged versions used in these studies were found to retain all the normal Gal80 regulatory functions (Jiang et al. 2009). At the outset, the central question driving this work was how does these clusters arise and what are the defining properties of these clusters. It was hypothesized that these Gal80 clusters most likely represent higher order self-assemblies of Gal80 (based on the number of

Gal80 molecules in each cluster) that are associated with Gal4 bound to UASGAL sites.

To test this central hypothesis, the frequency of colocalization between the Gal80-

2GFP clusters and the GAL1-10-7 locus containing six UASGAL sites on chromosome

II was determined and compared with the frequency of colocalization between the clusters and the LEU2 locus (no UASGAL sites). The GAL1-10-7 locus has the highest 85 number of UASGAL sites (six) in close proximity in the yeast genome. Gal80-2GFP was found to colocalize significantly more frequently with the GAL1-10-7 locus compared to with the LEU2 locus that has no UASGAL sites. The LEU2 locus did however show an unexpectedly high frequency of colocalization (44%). It is to be noted that both the LEU2 and the GAL1-10-7 loci are centromere linked (LEU2 linked to CEN3, GAL1-10-7 linked to CEN2). Therefore, one possible explanation for such a high frequency of colocalization for the LEU2 locus may be that centromere clustering (Jin et al. 2000) is bringing these two loci closer to each other and contributing to apparent higher than random colocalization frequency for the LEU2 locus to the Gal80 cluster. Therefore, one important future experiment would be to test the frequency of random colocalization of a non-UASGAL-containing locus that is not centromere linked (such as the HIS3 locus on chromosome XV).

Deletion analysis was used to ascertain if the UASGAL sites associated with the GAL1-

10-7 locus are required for the high frequency of colocalization. It was observed that deletion of all six UASGAL sites resulted in a decrease in colocalization frequency, suggesting that the association between a Gal80 cluster and the GAL1-10-7 locus is dependent on the UASGAL sites. Moreover, when the divergent region (DVR) containing the four UASGAL sites between the divergently transcribed GAL1 and

GAL10 genes was inserted into the LEU2 (no native UASGAL site) locus or into the

GAL3 locus (contains one native UASGAL site), a significant increase in association between each of these loci and a Gal80 cluster was noticed. Interestingly, it was observed that the association of a Gal80-2GFP cluster with the LEU2 locus 86 containing the inserted DVR increased even further and significantly when the four

UASGAL sites within the DVR of the native GAL1-10-7 locus on chromosome II were deleted. This response suggested that there might be competition between UASGAL sites at different loci for the Gal80 clusters. On the other hand, although deletion of all six UASGAL sites of the GAL1-10-7 locus resulted in a drastic loss of association of the locus with the cluster, deletion of either only the four UASGAL sites in the GAL1-

10 DVR of the GAL1-10-7 region or the two UASGAL sites in the GAL7 promoter region did not result in decrease in colocalization of the locus with a Gal80 cluster.

Thus retention of either the two UASGAL sites of the GAL7 promoter region or the four UASGAL sites of the GAL1-10 DVR region was sufficient for maintaining strong colocalization. Importantly, even though Gal80 clusters associated strongly with the

WT GAL1-10-7 locus and deletion of all six UASGAL sites significantly reduced this association, deletion of all six UASGAL sites did not alter the number of Gal80-2GFP cluster per nucleus. Taken together, these results demonstrate that Gal80 clusters nucleate at regions rich in UASGAL sites, and that there is probably competition amongst different UASGAL sites in the genome for Gal4-associated Gal80 (since Gal80 does not bind to DNA). It may well be that the GAL1-10-7 locus with six closely spaced UASGAL sites outcompetes the other sites for nucleation of Gal80 clusters, but in the absence of these six UASGAL sites, other UASGAL sites in the genome acquire increased access to Gal4 to nucleate Gal80 clusters. Such a scenario might explain why deletion of GAL1-10-7 results in no net change in the number of Gal80 clusters per cell. The observed correlative association of Gal80-2GFP clusters with the

UASGAL sites strongly suggested a role for Gal4 in the formation and/or maintenance 87 of clusters as Gal80 by itself does not bind to DNA (Lue et al 1987, Platt and Reece

1998) but does bind strongly to Gal4AD (Kd ~0.3-20 nM)) in vitro (Lue et al. 1987,

Wu et al. 1996, Melcher and Xu 2001). Accordingly, association of Gal80 clusters with the UASGAL was thought to be mediated by Gal4. This question could be addressed comparing the states of Gal80 clusters in the WT GAL4 and the gal4 deletion cells. However, to do so, it was first necessary that Gal80-2mYFP be expressed from a Gal4-independent promoter because Gal80 transcription itself is regulated by Gal4 (Shimada and Fukasawa 1985) and therefore deletion of Gal4 would reduce Gal80 levels and could affect Gal80 cluster formation. When Gal80-

2mYFP was expressed from the Gal4-independent STE50 promoter, it was observed that it made Gal80 clusters indistinguishable from those observed when Gal80-

2mYFP was expressed from GAL80 promoter in the presence of Gal4. In contrast, no cluster was observed in the absence of Gal4. This result clearly indicates that the occurrence of Gal80 cluster requires the Gal4 protein or protein/s dependent on

Gal4 activity.

Based on the above result, it was next asked what could be the role of Gal4 for Gal80 cluster formation. Because Gal4 binds specifically to UASGAL sites via its N-terminal

DBD and interacts with Gal80 through its C-terminal AD, it was hypothesized that it might be providing the conduit between UASGAL sites and Gal80 resulting in nucleation sites to form large Gal80 molecular assemblies built up by Gal80 self association. If Gal4 would be providing such a conduit through its DBD, it would be expected that the requirement of Gal4 could be bypassed if Gal4 DBD is fused to 88 Gal80 allowing Gal80 to bind UASGAL DNA directly. It was observed that in the presence of PGAL4-DBD-Gal80, Gal80-2mYFP expressed from its own promoter made clusters albeit slightly fainter than usual. On the other hand, when PGAL4-DBD-Gal80-

2mYFP alone was expressed in the absence of Gal4 and Gal80, it made typical clusters. These observations demonstrate that Gal80 self-association together with dependency of Gal80 clusters on Gal4 can be understood on the basis of Gal4’s ability to bind simultaneously with Gal80 and the UASGAL.

The above results led to the question as to whether PGAL4-Gal4-2mYFP/2GFP can make clusters. There have been no reports in the literature of visualizing Gal4 as a bright focus. This is understandable, as Gal4 is an extremely low abundance protein

(Laughon and Gesteland 1982). In fact others have had to use artificial constructs to target overproduced Gal4 to the nuclear membrane to visualize it as a dot

(Abramczyk et al. 2012). There has been one report of observing Gal4-Gal80 interaction using bimolecular fluorescence complementation where both the proteins were expressed from their genomic loci (Barnard et al. 2011). However, the strains used in this study showed growth impairment and whole cell extracts from these strains exhibited reduced (Gal1) activity (Barnard et al. 2011).

As mentioned previously, all the fluorescent protein-tagged proteins (Gal4 and

Gal80) retained functions similar to that of their untagged versions (Jiang et al.

2009). Also, none of the strains in this study showed any discernible growth defects.

In this work, when Gal4-2mYFP/2GFP was expressed from the GAL4 promoter on a low copy CEN vector, surprisingly, it was found to make a bright, visible focus. 89 Notably, it was observed that unlike Gal80 clusters, Gal4 foci did not dissipate in galactose. Moreover, Gal4 foci formation was not dependent on Gal80. Additionally, fluorescent intensity comparison between Gal4 foci in the presence and absence of

Gal80 did not show any significant difference. Taken together, these results suggest that Gal4 foci are different in nature and behavior from Gal80 clusters. Additionally, it was observed that Gal4-2GFP/2mYFP makes one bright focus and that this focus associates strongly with the GAL1-10-7 locus. Estimate of the number of molecules in the bright Gal4 focus (determined by photon counting) yielded a range of 20 to 60 molecules per focus with the average being 40. This number is higher than expected based on the published findings that Gal4 binds to UASGAL as a dimer (Silver et al.

1984, Bram and Kornberg 1985, Giniger et al. 1985, Bram et al. 1986, Ma and

Ptashne 1987b, Silver et al. 1988, Carey et al. 1989, Marmorstein et al. 1992). Thus one would expect just six dimers of Gal4 to be localized to the GAL1-10-7 locus and after the DNA replication and before the anaphase, there would be 12 dimers associated with these regions of the two sister chromatids (Uhlmann 2009 and references therein) (where six dimers would bind to the six UASGAL sites of the

GAL1-10-7 locus of each sister chromatid). It is tempting to speculate two mutually non-exclusive possibilities to explain the discord between the expected and the estimated (based on photon counting) number of Gal4 molecules associating with the GAL1-10-7 locus. One possibility would be that Gal4 might oligomerize beyond the dimer form. This could occur through the coiled coil motifs in Gal4 ((ExPasy

Bioinformatics Resource Portal; program: COILS; http://embnet.vital- it.ch/software/COILS_form.html, Carey et al. 1989, Marmorstein et al. 1992). 90 Another possibility which does not require Gal4 oligomerization, is that the bright

Gal4 foci observed here are nucleated at the UASGAL sites in GAL1-10-7 and through spatial proximity with Gal4 bound to other distant UASGAL sites the apparent number of closely colocalized Gal4 molecules is increased beyond 24. Yet, another possibility is that the photon counting method employed here overestimated the number of Gal4 molecules in the visible foci, but this seems unlikely to account for the observed large difference between the number of molecules in some of the Gal4 foci (60 molecules) and the maximum number of Gal4 molecules that theoretically can be expected to be associated with the GAL1-10-7 locus (24 molecules). It should be pointed out that there has been no evidence in the literature that Gal4 self- associates to form higher order oligomers other than dimers through its N-terminal dimerization domain (Carey et al. 1989, Marmorstein et al. 1992). However as discussed in the introduction (Chapter 1), Gal4 does have a predicted coiled-coil domain in the middle region (ExPasy Bioinformatics Resource Portal; program:

COILS; http://embnet.vital-it.ch/software/COILS_form.html) in addition to the strong coiled coil domain in the N-terminus (Carey et al. 1989, Marmorstein et al.

1992). Given the well-established roles of the coiled coil domains in protein oligomerization (Burkhard et al. 2001 and references therein), the possibility of

Gal4 participating in higher-order self-assemblies remains open. One can speculate that higher order assemblies of Gal4 molecules around GAL1-10-7 may potentially allow a low abundance protein like Gal4 to activate GAL1 transcription rapidly upon induction and maintain strong activation even under the condition where active

Gal4 is targeted for proteasomal degradation (Muratani et al 2005). 91

One trivial explanation for the observed Gal80-2mYFP/2GFP clusters and Gal4-

2mYFP/2GFP foci could be that we are merely observing the interaction of Gal80 and Gal4 in their established 2:2 stoichiometry (Kumar et al. 2008, Melcher and Xu

2001) at the UASGAL sites. However, there are several lines of evidences in this work that are not consistent with this idea. Firstly, the photon counting estimates of the number of molecules for Gal80 and Gal4 clusters are not similar, with Gal80 clusters showing a much higher range compared to Gal4 foci. Secondly, when the six UASGAL sites at the GAL1-10-7 locus were deleted, the bright Gal4 focus that colocalized to that locus almost completely disappeared and yet, under the same condition where association of a Gal80 cluster with this locus was lost, other Gal80 clusters persisted. Thirdly, the number distributions for Gal4 foci and Gal80 cluster were not similar in the presence of all six UASGAL sites in the native GAL1-10-7 region and were very different in the absence of it (discussed in more detail in the next paragraph). Finally, when Gal4 foci and Gal80 clusters were observed simultaneously, there were quite a few instances where the Gal4 foci and Gal80 clusters did not completely colocalize, and this difference was significantly enhanced upon deletion of all six UASGAL sites in the GAL1-10-7 region. All of these considerations point to the possibility that even though Gal80 clusters depend on interaction of Gal80 with the UASGAL-bound Gal4, the interaction appears not to be stoichiometric. It appears that there could be more Gal80 molecules present in the visible Gal80 clusters than Gal4 molecules in the Gal4 foci. Additionally, if we only consider the number of UASGAL sites and the maximum number of Gal4 and in turn 92 Gal80 that can associate with them through a 2:2 stoichiometric binding, the number of Gal4 and Gal80 molecules associating with these UASGAL sites should have been much less than what we observe, as mentioned before. Therefore, the long-held picture of one UASGAL site bound by one Gal4 dimer that is bound by one

Gal80 dimer (Silver et al. 1984, Bram and Kornberg 1985, Giniger et al. 1985, Bram et al. 1986, Ma and Ptashne 1987b, Silver et al. 1988, Carey et al. 1989,

Marmorstein et al. 1992, Melcher and Xu 2001, Kumar et al. 2008) cannot explain the presence of such large number of Gal4 and Gal80 molecules at the GAL1-10-7 locus. The possible occurrence of such large molecular assemblies of both Gal4 and

Gal80 molecules associated with the UASGAL sites within the GAL1-10-7 locus warrants further investigations, as they may reflect yet undiscovered mechanisms in the regulation of GAL genes.

One rather unexpected result in this work is that in-spite of strong evidence for the association of both the Gal80 cluster and the Gal4 focus with the UASGAL sites of

GAL1-10-7, the deletion of all six UASGAL sites had very different effect on them.

Gal80 clusters did not show any change in the number distribution upon deletion whereas bright Gal4 focus almost completely disappeared. One hypothesis that could reconcile this apparent discrepancy is the fact that there are approximately

18-30 UASGAL sites throughout the yeast genome (Ren et al. 2000, TRANSFAC, SCPD,

Rhee and Pugh 2011, Wang et al. 2011). Therefore, in the cells lacking six UASGAL sites of the GAL1-10-7 locus, Gal80 clusters may well nucleate off of the remaining sites through extensive Gal80 self-associations. The experiment in this work that 93 revealed competition between different UASGAL sites for the Gal4-associated Gal80 is consistent with this possibility as previously discussed. Once nucleated by

UASGAL-bound Gal4, Gal80 molecules in the newly nucleated clusters can associate with free, non-Gal4 bound, Gal80 molecules and/or Gal80 molecules of other clusters to extend the higher order assembly independent of further direct binding of Gal80 to Gal4. This notion is consistent with several observations in the literature of Gal4-independent self-association of Gal80, both in vitro and in vivo

(Melcher and Xu 2001, Pilauri et al. 2005, Lavy et al. 2012, Egriboz et al. 2013,).

Also, in this work it was observed that when fused to Gal4 DBD, Gal80 could make typical clusters even in the absence of Gal4. Thus, it appears that Gal80 requires

UASGAL–bound Gal4 to nucleate the cluster but then can form higher order self- assemblies independent of direct interaction with UASGAL-bound Gal4. On the other hand, it seems that Gal4 needs close physical proximity of a number of UASGAL sites to form a visible focus possibly through one or more of the mechanisms discussed previously in this section.

The discovery of large molecular assemblies of Gal80 raised the question as to whether these might span or bridge between two or more distinct, distant Gal4-

UASGAL complexes. An attempt was made to test this idea by measuring the distances between the two GAL1 loci (each containing six UASGAL sites) in a diploid and then comparing that with the distances between a GAL1 (six UASGAL sites) and a

LEU2 (with no UASGAL site) locus to see if there is any significant statistical difference in distances between the two sets of loci. Given the fact that both the 94 GAL1 and the LEU2 are centromere linked as mentioned previously, nocodazole was used to reduce the possible effect of centromeric clustering on the distances between these two loci (Jin et al. 2000). This experiment did not reveal any evidence that the two GAL1 loci are significantly closer to each other than a GAL1 and a LEU2 locus. Thus, it remains unclear if indeed there is any bridging between different

UASGAL sites. However, finding evidence for bridging is challenging for two reasons.

Firstly, there are many other UASGAL sites in the genome (Ren et al. 2000,

TRANSFAC, SCPD, Rhee and Pugh 2011, Wang et al. 2011) and it is quite possible that a given ensemble of Gal80 molecules (a cluster or part of a cluster) might undergo exchange to the effect of constituting bridging between different combinations of UASGAL sites from time to time. Such a transient nature of a cluster may arise from the dynamic nature of the Gal80-Gal80 associations that are presumably defining them (Figure 3.1B). Therefore, the method adopted in this work may not capture this transient association. Chromosome conformation capture or 3C (Dekker et al. 2002) is a method that is commonly employed for determining long range chromosomal interaction, and in future studies such an approach could be applied to get a more definitive answer to this question.

A major remaining question regarding Gal80 cluster and Gal4 foci is whether there is any functional significance of these clusters? Given the importance of self- association in Gal80 functioning (Melcher and Xu 2001, Pilauri et al. 2005, Egriboz et al. 2013,) and the presence of such large number of Gal80 molecules in the Gal80 clusters and their associations with the GAL1-10-7 locus, it was hypothesized that 95 these clusters are providing tighter repression (repressosome hypothesis) (see the gal operon work of Aki and Adhya 1997, Lewis and Adhya 2002). This idea was tested in this work by using a system originally developed in the Singer lab (Larson et al. 2011), whereby one can visualize newly synthesized transcript in real-time at a given locus as described in the results section. Finding a positive correlation between the Gal80 cluster dissipation or noncolocalization with the locus under study and active transcript production would be consistent with the repressosome hypothesis. To this end, 103 cells were randomly selected for analysis after exposing them to a low-level induction stimulus (0.05% galactose) for 20 minutes. This analysis showed that the cells could be largely grouped into three categories based on their response. One category of cells showed the Gal80 cluster colocalizing to the assayed GAL gene locus and no transcript dot was visible. The second group showed only transcript dot but no co-localizing Gal80 cluster. The third group showed neither Gal80 cluster nor any transcript dot. Most importantly, less than 2% cells showed both the Gal80 cluster associating with the locus and the presence of a visible transcript dot. This preliminary result is encouraging but cannot by itself be taken as evidence that Gal80 clusters represent sites of very tight repression (that persist under weakly inducing conditions). Larger sample sizes along with time- lapse movies are needed to get a clear answer to this question particularly because there were about 20% cells with no visible clusters and yet they did not show any transcript. Such cells might represent stochastic events that limit capacity to transcribe (for example, no Gal4 at the UASGAL site) (reviewed in Kærn et al. 2005).

It should be noted that an RNA FISH-based (fluorescence in-situ hybridization) 96 single cell analysis of galactose induction of GAL1 transcription showed that even under normal inducing (2% galactose) condition only two-thirds of the cells showed visible transcript (Cabal et al. 2006). An obvious approach to investigate this further would be to check if lack of transcript formation in the absence of Gal80 cluster is due to the absence of Gal4 at the locus. This would require one to be able to see both the Gal4 and Gal80 at the same time and monitor the relationship between the interaction/colocalization between them and the formation or lack of formation of transcript. An alternative explanation for the occurrence of cells that showed neither the transcript dot nor the Gal80 cluster that is associated with the marked locus under weakly inducing conditions (0.05% galactose) is that Gal3 under such conditions may be capable of impairing Gal80 self-association (dissipating Gal80 clusters) but less or not capable of dislodging a single dimer of Gal80 from Gal4. This hypothesis is warranted by the published results that in vitro Gal80 dimerizes with strong affinity (Kd = 0.1-0.3 nM) but tetramerizes with moderate affinity (Kd = 50 nM) (Melcher and Xu 2001) and moreover, a dimer of Gal80 binds more strongly to a dimer of Gal4AD (Kd ~ 0.3-20 nM) ((Lue et al. 1987, Wu et al. 1996, Melcher and

Xu 2001) than to another Gal80 dimer (Kd = 50 nM) (Melcher and Xu 2001).

Therefore, under weakly inducing condition, Gal3 might be able to disrupt weak interactions between Gal80 molecules that give rise to these cluster, but may not be capable of disrupting strong association between a Gal80 dimer and a Gal4 dimer and thus the GAL gene would still be repressed even in the absence of any associated cluster.

97

Overall, in this work concerning Gal80 clusters and Gal4 foci, several observations have been made, notable amongst those are: a, the occurrence of large molecular assemblies of Gal80 which is referred to as clusters; b, reformation of these cluster upon galactose depletion in the absence of synthesis of any new Gal80 protein; c,

UASGAL site-dependent associations of these clusters with the GAL1-10-7 locus, d, suggestive evidence for competition among different UASGAL sites for Gal80 clusters; e, non-dependence of Gal80 cluster number distributions within a population on the presence of six UASGAL sites in the GAL1-10-7 locus; f, Gal4-dependent occurrence of

Gal80 clusters. Additionally, Gal4-2GFP/2mYFP expressed from the native GAL4 promoter has been visualized as bright foci associated with the native GAL1-10-7 locus on chromosome II. This as far as we know, is a novel observation.

Furthermore, Gal80 and Gal4 were observed simultaneously in a single cell under microscope. Although some insights about Gal80 clusters as well as Gal4 foci have been gained, there are still several unanswered questions, which would guide any future efforts. Some of the questions are related to Gal4 foci such as what part/region of Gal4 is important for formation of such visible foci of Gal4? Does the middle region of Gal4 participate in higher order oligomer formation? What is the role of the N-terminal DBD, or the C-terminal AD of Gal4 in formation of these foci?

Can Gal4 that is mutationally impaired in transcription activation function make such foci? For Gal80 clusters, the issue of requirement for self-association still remains unresolved because of lack of any suitable mutant that can effectively uncouple self-association function of Gal80 from Gal4 binding. To understand the 98 functional relevance of Gal80 clusters, further investigation is needed. In particular we need to further pursue the hypothesis of a Gal80 repressosome. The issue of bridging between different UASGAL sites should also be revisited with different methods such as chromosome conformation capture. To summarize, these investigations of Gal80 clusters have answered some questions but have left many more unanswered. Further studies of Gal80 clusters are needed to gain further insights into the extensively studied, but seemingly increasingly complex GAL genetic switch.

99

Figure 3.1: Gal80-2GFP exhibits predominantly 1, 2, or 3 intranuclear clusters:

A. Sc862 cells with Gal80 tagged at the 3’ end in frame with 2GFP shows predominantly 1, 2, or 3 intranuclear clusters. Cells were grown in 3% glycerol-2% lactic acid media to mid log phase before imaging. H2B1-mCherry (pFJ35) is used as nuclear marker. Arrow indicates Gal80-2GFP clusters.

B. Time course showing the variability in terms of the size, shape and intensity of

Gal80-2GFP clusters in the strain Sc862. Cells were grown in 3% glycerol-2% lactic acid media to mid log phase before imaging Images were taken at an interval of 15 seconds.

For both A and B, images are maximum intensity projection of 11 Z-stacks with the Z spacing of 0.4 μm. Arrow indicates cluster. Scale bar 1μm.

100

Figure 3.1:

A.

No. of Gal80-2GFP H2B1-mCherry Composite DIC Clusters

1

2

3

B. Gal80-2GFP

O Sec 15 Secs 30 Secs

45 Secs 60 Secs 75 Secs

Gal80-2GFP

101

Figure 3.2: Gal80-2GFP clustering is observed in raffinose but not in glucose:

Sc862 cells expressing Gal80-2GFP from its genomic locus is grown in either 2% glucose or 2% raffinose to mid log phase before imaging.

A. When grown in glucose Gal80-2GFP shows very faint or no clusters.

B. In raffinose, Gal80-2GFP makes typical cluster as seen in glycerol-lactic acid media.

H2B1-mCherry (pFJ35) used as a nuclear marker. Images are maximum intensity projection of 11 Z-stacks at spacing of 0.4 μm. Arrow indicates Gal80-2GFP cluster.

Scale bar 1 μm.

102

Figure 3.2. A.

Gal80-2GFP H2B1-mCherry Composite DIC

In glucose

B.

In raffinose

103

Figure 3.3: Preexisting Gal80-2GFP can reassemble into cluster upon galactose depletion: To establish that cycloheximide is effective in blocking protein synthesis for the entire duration of the experiment (2 hours) following experiments (A and B) were carried out. A. Cells of the strain Sc854 (endogenous GAL1 tagged at its 3’end in frame with 2GFP) were grown in 3% glycerol and 2% lactic acid to early to mid log phase and either cycloheximide (CHX) to final concentration of 25 25μg/ml or only DMSO (Dimethyl sulfoxide) was added to the cells and the cells were incubated for 30 minutes and then galactose was added to the cells to final concentration of

2% and total protein was extracted from the cells either just before adding the galactose (0 hour) or after 1 hour, 2 hours and 3 hours after addition of galactose.

Immunoblotting was used to detect the total amount of Gal1-2GFP protein under different experimental conditions. Alpha tubulin was used as a loading control. B.

PGAL1-2GFP (pOE33) was expressed from a low copy CEN vector in the presence of

PGAL4-Gal4 (pBM292) expressed from another low copy CEN vector in sc745 (Δgal4).

Cells were grown in 3%glycerol 2%lactic acid media to early to mid log phase and either CHX to final concentration of 25μg/ml or only DMSO was added to the cells and cells were incubated for 30 minutes and then galactose was added to the cells to final concentration of 2%. Images were taken either just before adding galactose or

1 hour, 2 hours, and 3 hours after galactose addition. (C-F) Sc862 expressing Gal80-

2GFP (isogenic to Sc854 except GAL80 instead of GAL1 was tagged in-frame at its 3’ end with 2GFP) from its genomic locus was grown to early to mid log phase at which time cycloheximide was added to final concentration of 25μg/ml (same 104 concentration as used for determining the effectiveness of cycloheximide for the duration of the experiment described in A) and incubated for 30 min before adding galactose to final concentration of 2%. After one-hour incubation, cells were washed twice and resuspended in glycerol lactic media with no galactose and fresh cycloheximide (25μg/ml final concentration). Images are taken at following time points:

C. Immediately prior to addition of galactose.

D. After 1 hour incubation in 2% galactose.

E. One hour after galactose removal (- galactose). Gly/lac (3%glycerol, 2% lactic acid)

F. Two hours after galactose removal (- galactose). Images are maximum intensity projection of 11 Z-stacks with Z-spacing of 0.4µm. Arrows indicate Gal80-2GFP cluster. Scale bar 1 µm.

+CHX = with cycloheximide 25μg/ml

-CHX = only DMSO hrs = hours

105 Figure3.3:

A.

-CHX +CHX

+Galactose 0 hr 1hr 2hrs 3hrs 0hr 1hr 2hrs 3hrs

Gal1-2GFP

α-tubulin

B.

PGAL12GFP DIC

-CHX

+CHX

In gly/lac

continued…

106 …Figure 3.3 continued

PGAL12GFP DIC

-CHX

+CHX

+Galactose 1 hour

PGAL12GFP DIC

-CHX

+CHX

+galactose 2 hours

continued..

107 …Figure 3.3 continued

PGAL12GFP DIC

-CHX

+CHX

+galactose 3 hours

C. Gal80-2GFP DIC

In gly/lac

continued..

108 …Figure 3.3 continued

D. Gal80-2GFP DIC

+Galactose 1 hour E.

Post wash gly/lac 1 hour (minus galactose) F.

Post wash gly/lac 2 hours (minus galactose)

109

Figure 3.4: Gal80 clusters colocalize with the GAL1-10-7 locus:

A. General scheme for assessing colocalization. Insert LacOx64 array

downstream of the locus to be tested for colocalization with Gal80-2GFP

cluster. Binding of LacI-mCherry (pME8) will mark this locus as a red dot. B.:

Schematic depiction of the GAL1-10-7 region on ChrII. C. Schematic

representation of the construct used in this study for establishing true

colocalizaton standard. The P -GSTx 8 containing array of 8 tandem GAL1

repeats of GST gene under GAL1 promoter inserted around 9.7 kb away from

LacOx64 array that contains 64 tandem repeats of Lac operator sequences,

(From Jiang et al. 2008). D. Strain Sc913 containing both the P -GSTx 8 GAL1

tandem repeat array as well as 2GFP tagged (at 3’ end) Gal80 and LacI-

mCherry was grown to mid log phase before image acquisition. One

representative image of maximum intensity projection of 11 Z-stacks was

shown to illustrate true colocalization. Scale bar 1μm. In the lower panel the

same image but of a single Z-plane was blown up 32 times. E. Graphical

representation of the result of line scans performed in ImageJ

(http://imagej.nih.gov/ij/) for intensity profile performed on the image

shown in the lower panel of 4D. 4F. The table at the bottom shows

coordinates of different pixels. For true colocalization between two

fluorescent dots (say green and red), if the highest intensity pixel for one (say

green) fluorescent dot is at position (0,0) then highest intensity pixel for true

110 colocalization for the other fluorescent dot (red) according the standard I set

has to be in one of the nine pixels.

111

Figure 3.4. GAL gene LacOx64 A.

UASGAL

B LacOx6 GAL7 GAL10 GAL1 4 Chr II

Divergent Region UAS 6 GAL4 Binding(DVR) Not drawn G sites to scale

Native Chromosome II, showing UASGAL-rich GAL1-GAL10-GAL7 region

C. P -GSTx 8 GAL1 LEU2 LacOx64 locus 4

9.7 kb 32 UASGAL sites

Standard for colocalization

continued…

112 …Figure 3.4 continued

D. Images acquired at 100x Gal80-2GFP LacI-mCherry Composite DIC

1x

32x

E.

Gal80-2GFP

LacI-mCherry

FLUORESCENCE INTENSITY A.U. ! Pixel

F.

-1,1, 0,1, 1,1,

-1,0, 0,0, 1,0,

-1,-1, 0,-1, 1,-1,

113 Figure 3.5: Gal80 clusters colocalize with the GAL1-10-7 locus: Colocalization frequency between Gal80-2GFP cluster and different genomic loci marked by LacI- mCherry (pME8) dot (binding to the LacOx64 array inserted into the locus under investigation) was determined. Statistical comparisons were done using comparison between two binomial proportions with normal approximation. Relevant genotypes for these otherwise isogenic strains are provided next to the small square boxes of the same color as that of the bar on the chart. A. Shows the effect of deletion of different UASGAL sites in GAL1-10-7 locus. All the comparisons (in A) were done with frequency of colocalization of WT GAL1-10-7 locus with Gal80-2GFP clusters and p values are shown at the top of the bar of each strain that was compared with B.

Effect of insertion of the GAL1-10 DVR into ectopic locus. Brackets indicate the pair used for the pair-wise comparison, the p value of the comparison is shown right below the respective brackets. LEU2 locus without any UASGAL sites was used to determine the frequency of random colocalization. Frequency of colocalization is shown next to the respective bar. Image acquisition and analysis ware done blind.

Analysis was done using the standard as depicted in Figure 3.4. Strains used are

Sc1020, Sc1031, Sc1032, Sc1041, Sc1044, Sc1050, and Sc1082, Sc1084. The relevant genotypes mentioned in the result section and also provided in Table 3.8. Error bars are ±SDs (standard deviations).

114 Figure 3.5.

A.

1 p<0.0001 0.3735 0.0278 <0.0001 0.83 0.9 0.78 80 clusters 0.8 0.89 0.7 0.6 0.44 0.5 0.29 0.4 0.3 0.2 0.1 0

Fraction of cells colocalizing with GAL No of UASgal 0 6 4 2 0

sites

LEU2 GAL1-10-7 ΔPGAL1-10 GAL1-10-7 WT

GAL1-10-7 ΔPGAL1-10 + ΔPGAL7 GAL1-10-7 ΔPGAL7

continued…

115

…Figure 3.5 continued

B.

! No of 0 4 4 1 5 6 UASGAL sites

--2 LEU2 GAL3

LEU2 +DVR GAL3 +DVR

LEU2 +DVR GAL1-10-7 + ΔPGAL1-10 WT

116

Figure 3.6. Deletion of UASGAL sites in GAL1-10-7 does not affect the number distribution of Gal80-2GFP cluster in yeast population: Number of Gal80-2GFP clusters were counted in 3 independent groups of randomly selected 100 cells (total

300 cells for each strain) for each strain. The data is presented as percentage of cells with a particular number of clusters in the sample population. Strains used are

Sc1020 and Sc1031; the relevant genotypes are provided in the result section and also provided in Table 3.9. Two-tailed Student’s t-test was carried out to compare different distributions and obtain the p values. All p values are greater than 0.05.

Error bars are ±SDs.

117 Figure 3.6.

45

40

35

30

25

20

15 Percentage of cells 10

5

0

1 2 3 4 or more Number of Gal80-2GFP clusters

WT GAL1-10-7

ΔGAL1-10-7

118

Figure 3.7: Gal80-2mYFP cluster formation is dependent on Gal4: Sc732

(Δgal80, Δgal4) expressing PSTE50-Gal80-2mYFP (pSG45) from a CEN vector was grown either in the absence of Gal4 (7A)(empty vector pRS414) or in the presence of Gal4 (7B) (pBM292-expressing Gal4 from its own promoter in CEN vector) in

3%glycerol-2%lactic acid media to mid log phase before image acquisition. H2B1- mCherry (pFJ35) was used as nuclear marker. Images are maximum intensity projections of 12 Z stacks with the Z-spacing of 0.4μm. Arrow indicates Gal80-

2mYFP cluster. Scale bar 1μm

119 Figure 3.7.

A. Δgal80Δgal4 Gal80-2mYFP H2B1-mCherry Composite DIC

B.

Δgal80WTGAL4

120 Figure 3.8: Requirement of Gal4 for Gal80-2mYFP cluster formation can be bypassed by providing Gal80 with of Gal4DBD: Sc1068 expressing Gal80-2mYFP

(Δgal4) from its genomic locus was transformed with either A. PGal4 GAL4DBD-Gal80

(pEB1) or B. PGal4 GAL4DBD-Gal80-2mYFP (pEB2) expressed from CEN vector were grown to mid log phase before image acquisition. Nup49-mCherry (pFJ152) was used as nuclear marker. Arrows indicate Gal80-2mYFP clusters. Images are maximum intensity projections of 12 Z sections with the Z-spacing of 0.4μm. Scale bar 1μm.

121 Figure 3.8

A. +PGal4 GAL4DBD-Gal80

Gal80-2mYFP Nup49-mCherry Composite DIC

B. +PGal4 GAL4DBD-Gal80-2mYFP

122 Figure 3.9: PGal4 DBD-Gal80-2mYFP can make intranuclear cluster in the absence of both Gal4 and Gal80 but SV40 NLS–Gal80-2mYFP cannot: Sc1153

(Δgal80, Δgal4, Sec63-mCherry) cells transformed with A. PGal4 DBDGal80-2mYFP

(pEB2) or B. PGal4 DBDL32P Gal80-2mYFP (pSG47) or C. PGal4 SV40NLS Gal80-2mYFP were grown to mid log phase in 3%glycerol-2% lactic acid media before image acquisition. Endogenous Sec63 tagged with mCherry in frame at the 3’ end used as nuclear marker. Images are maximum intensity projections of 12 Z sections with the

Z-spacing of 0.4μm. Arrow indicates Gal80-2mYFP cluster. Scale bar 1μm.

123 Figure 3.9.

A. PGal4 Sec63-mCherry Composite DIC DBDGal80-2mYFP

B. PGal4 Sec63-mCherry Composite DIC DBD L32P Gal80-2mYFP

C. PGal4 Sec63-mCherry Composite DIC SV40NLS Gal80-2mYFP

124 Figure 3.10: Gal80 mutant Gal80N230R defective in dimerization does not make cluster: Sc725 (Δgal80) expressing either A. PSTE50-Gal80N230R-2mYFP

(pSG46) or B. PSTE50-Gal80-2mYFP (pSG45) from CEN vector were grown to mid log phase in 3%glycerol-2%lactic acid media before image acquisition. H2B1-mCherry was used as nuclear marker. Images are maximum intensity projections of 12 Z sections with the Z-spacing of 0.4μm. Scale bar 1μm.

125 Figure 3.10.

A. Gal80N230R

Gal80-2mYFP H2B1-mCherry Composite DIC

B. WT Gal80

126

Figure 3.11: Gal4-2GFP appears as intranuclear foci both in the absence or presence of galactose: Sc745 (Δgal4) expressing Gal4-2GFP from its own promoter from a CEN vector was grown in 3%glycerol 2%lactic media and images were acquired either without galactose A with 2% galactose for 1 hour B. H2B1-mCherry

(pFJ35) was used as nuclear marker. Images are maximum intensity projections of

12 Z sections with the Z-spacing of 0.4μm. Arrows indicate Gal4-2GFP foci. Scale bar

1μm.

127 Figure 3.11.

A. Without galactose

Gal4-2GFP H2B1-mCherry Composite DIC

B. With galactose for 1 hr.

128

Figure 3.12. Gal4 foci formation is not dependent on Gal80. Fluorescent intensities of the Gal4-2mYFP foci were measured both in the presence (Sc1160) and absence (Sc1161) of Gal80 (n= 50) as described in the material and method section. Two-tail Student’s t test was performed which showed no significant difference in the fluorescent intensities of Gal4-2mYFP in the presence versus in the absence of Gal80. Error bars are ±SDs.

129 Figure 3.12.

Intensity AU Fluorescence

!

Δ gal80 WTGal80

130 Figure 3.13: The brightest Gal4-2GFP focus colocalizes with the GAL1-10-7 locus: Sc1163 expressing Gal4-2GFP from its genomic locus was observed along with GAL1-10-7 locus marked by LacI-mCherry dot in A. WT GAL1-10-7 (Sc1163) or

B. All six UASGAL sites were deleted (Sc1206). Cells were grown in 3% glycerol-2% lactic acid to mid log phase before imaging. Images are maximum intensity projections of 12 Z sections with the Z-spacing of 0.4μm. Scale bar 1μm.

131 Figure 3.13.

A. WT-GAL1-10-7

Gal4-2GFP LacI-mCherry Composite DIC

B. ΔGAL1-10-7

132 Figure 3.14: Gal80 cluster and Gal4 foci can be simultaneously observed in the same cell: Sc1165 expressing Gal4-2GFP and Gal80 tdTomato from their genomic loci.

A. WT GAL1-10-7 (Sc1165) cells were grown to mid log phase in 3%glycerol- 2% lactic acid before imaging. B: Same strain grown in 3%glycerol- 2%lactic acid media to early to mid log phase and then galactose was added to final concentration of 2% and incubated for 2 hours before imaging. C. Sc1205, same strain as Sc1165 except the GAL1-10-7 locus with 6 UASGAL sites deleted. Images were acquired the same way as described above. Arrows indicate foci. Images are maximum intensity projections of 12 Z sections with the Z-spacing of 0.4μm. Scale bar 1μm.

133 Figure 3.14.

A. In glycerol-lactic acid media; WT GAL1-10-7

Gal4-2GFP Gal80-tdTomato Composite DIC

B. In galactose media; WT GAL1-10-7

C. In glycerol-lactic acid media; ΔGAL1-10-7

134

Figure 3.15: Schematic depiction of general scheme/approach to visualize two loci simultaneously as two fluorescent dots of different fluorescent proteins in the cell: Two different operator arrays are inserted into specific loci of interest in two haploid cells of opposite mating type. After mating and formation of diploid, the two loci of interest can be visualized as fluorescent dots upon binding of the cognate repressors tagged with fluorescent protein to these different operator arrays. In this experiment, Tet operator and TetR-GFP (Tet repressor-GFP) and Lac operator and

LacI-mCherry have been used.

Gal Gene locus i Gal Gene locus j

Mating MATa MATα LacOx64 TetOx512

haploid X haploid

TR-GFP a/α diploid

LacI-mCherry

135

Figure 3.16: Scheme showing the general strategy to observe real-time synthesis of transcript at a given locus. Method was developed in Singer lab

(Larson et al. 2011)

PP7-CP binding sequence 2CFP

mCherry PP7- C

Pol II LacI -

LacOx64 PGAL -GALGENE-PP7bsx24

136 Figure 3.17: Simultaneous monitoring of Gal80-2mYFP clusters and the transcription status of a given locus. Strain Sc1198 carries PGAL1-GST-PP7 inserted at the LEU2 locus close to the already inserted LacOx64 array. Gal80-

2mYFP is expressed from its genomic locus. Cells were grown in 3% glycerol -

2%lactic acid to mid log phase, then galactose was added to final concentration of

0.05% and after 20 minutes images were acquired. Transcripts carrying 24 PP7 binding sites are complexed with PP7-2CFP and will yield a visible dot. A. Cells were grown in 3%glycerol-2%lactic acid media before imaging. B. Galactose was added to

0.05% final concentration and incubated for 20 minutes before imaging. It shows a cell with cluster colocalizing to the LEU2 locus and there is no transcript. 32% of the cells out 103 randomly selected cells exhibited this. C. Showing cells with either no cluster-no transcript foci (white arrow -19%) or with transcript and no cluster

(colored arrow 47%). In each instance images of 12 Z-stacks were taken at 0.4μm Z spacing. A single Z slice is shown in all three images. Scale bar 1μm.

137 Figure 3.17.

A. In glycerol-lactic acid media

Gal80-2mYFP LacI-mCherry PP7-2CFP Composite DIC

B. In 0.05% galactose for 20 mins

C. In 0.05% galactose for 20 mins

138

Table 3.1:

Distribution of percentages of cells with a specific number of Gal4- 2GFP/2mYFP foci per nucleus. (N = 100)

GAL4 TAG GAL80 % of cells % of cells % of cells % of cells with 1 dot with 2 dots with 3 with 4 dots dots 2mYFP WT 32 33 32 3 2mYFP Δ 35 35 23 7 2GFP WT 54 31 15 2GFP Δ 49 36 14 1

139 Table 3.2:

Analysis of colocalization between Gal4-2GFP foci and Gal80-tdTomato cluster in the WT GAL1-10-7 strain.

Feature No. of cells/109 cells % (approximate) C 59 54 C+80 18 16.5 C+4 3 2.8 80 only 13 12 4 only 9 8.3 4 – 80 4 3.7 N 3 2.8

Table 3.3: Analysis of colocalization between Gal4-2GFP foci and Gal80-tdTomato cluster in the ΔGAL1-10-7 strain.

Feature No. of cells/100 cells % C 19 19 C+80 6 6 C+4 1 1 80 only 49 49 4 only 1 1 4-80 2 2 N 22 22

Abbreviations used in Table 3.2 and Table 3.3: C – Complete colocalization of Gal80-tdTomato cluster and Gal4-2GFP foci – equal number

C+80—Colocalization with additional Gal80-tdTomato cluster not associated with any Gal4-2GFP foci i.e. higher number of Gal80-tdTomato cluster than Gal4-2GFP

C+4 – Colocalization with additional Gal4-2GFP foci not associated with any Gal80tdTomato foci i.e. higher number of Gal4-2GFP foci than Gal80-tdTomato cluster

80 only – Only Gal80-tdTomato cluster detectable – no Gal4-2GFP foci

4 only – Only Gal4-2GFP foci detectable – no Gal80-tdTomato cluster

4-80 – Both Gal80-tdTomato cluster and Gal4-2GFP foci visible but they are not colocalizing

N- Neither Gal80-tdTomato nor Gal4-2GFP foci visible 140 There is no significant difference between the distances between two GAL1 loci and one GAL1 locus with LEU2 locus: Three-dimensional distances between the centroids of the two tagged loci as described, were measured in 100 cells and the mean and standard deviations (Stdev) were determined. The Kolmogorov-

Smirnov (KS) test was used to compare between any two distributions. The Results are presented in the tables below. Table 3.5 and 3.6 are results obtained with no nocodazole treatment (only DMSO) and Table 3.7 and 3.8 shows the result post nocodazole treatment.

Table 3.4: Without nocodazole treatment Locus1 Locus 2 Mean Distance Stdev n between the (nm) centroids (nm) LEU2 LEU2 628 499 100 LEU2 GAL1 701.4 364 100 GAL1 GAL1 605.6 268 100

Table 3.5: Comparisons between P value from KS test LEU2/LEU2 –LEU2/GAL1 0.016 LEU2/LEU2 – GAL1/GAL1 0.437 LEU2/GAL1 – GAL1/GAL1 0.039

Table 3.6: With nocodazole treatment Locus1 Locus 2 Mean Distance Stdev n between the (nm) centroids (nm) LEU2 LEU2 752 291 100 LEU2 GAL1 653.1 232 100 GAL1 GAL1 651.6 244 100

Table 3.7 Comparisons between P value from KS test LEU2/LEU2 –LEU2/GAL1 0.072 LEU2/LEU2 – GAL1/GAL1 0.035 LEU2/GAL1 – GAL1/GAL1 0.954

141 Table 3.8: Distribution of cells with respect to cluster colocalization to the

LacOx64 array and appearance of transcript dots. The strain Sc1198 where

PGAL1-GST-PP7 was inserted in the LEU2 locus close to the already inserted LacOx64 array and expressing Gal80-2mYFP from its genomic locus were grown in glycerol lactic acid to mid log phase, then 0.05% galactose was added and after 20 minute incubation, images were acquired. From the acquired images transcriptional status of PGAL1-GST-PP7 and the Gal80-2mYFP cluster colocalization to the locus (marked by LacI-mCherry dot) where PGAL1-GST-PP7 had been inserted were determined in

103 cells and the observations are tabulated in this table below:

Feature No. of observations/103 % (approximate) cells Gal80-2mYFP cluster 33 32 only Transcript foci only 48 47 Cluster + Transcript 1 1 Weak/diffused cluster + 1 1 Transcript Neither cluster nor 20 19 Transcript

142 Table 3.9:

Yeast strains used in this study

Strain No. Genotype Source Sc725 MATa ade1 ile leu2-3, 112 ura3-52 Blank et al. 1997 trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 gal80-ΔBglII Sc732 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 gal80-ΔBglII gal4Δ::LEU2 Sc745 MATa ade1 ile leu2-3, 112 ura3-52 Egriboz et al. trp1-HIII his3-Δ1 MEL1 2013 LYS2::GAL1UAS-GAL1TATA-HIS3 gal4Δ::LEU2 Sc841 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 GAL80-2GFP-NAT Sc854 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 GAL1-2GFP-KAN Sc862 MATa ade1 ile leu2-3, 112 ura3-52 Jiang et al. 2009 trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 GAL80-2GFP-NAT Sc913 MATa ade1 ile leu2-64xLacO-LEU2 Jiang et al. 2009 ura3-52 trp1-HIII his3-Δ1 MEL1 GAL80-2mCitrine –CaURA3 Sc1020 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal80-2GFP::NAT GAL1::LacOx64::LEU2

Sc1021 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal80-2GFP::NAT GAL1::LacOx64::LEU2 PGAL1Δ::KAN Sc1031 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-delta1 Gal80- 2GFP::NAT GAL1::LacOx64::LEU2 PGAL1Δ::KAN PGAL7Δ::SpHis5 continued… 143 …Table 3.9 continued

Sc1032 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-delta1 Gal80- 2GFP::NAT GAL1::LacOx64::LEU2 PGAL7Δ::SpHis5 Sc1041 MATa ade1 ile leu2-64xLacO- This study LEU2::UASGAL1-10-Kan ura3-52 trp1-HIII his3-Δ1 MEL1 Gal80- 2GFP::NAT Sc1044 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-delta1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 GAL80-2GFP-NAT GAL3::LacOx64::LEU2 Sc1050 MATa ade1 ile leu2-64xLacO- This study LEU2::UASGAL1-10-Kan ura3-52 trp1-HIII his3-Δ1 MEL1 Gal80- 2GFP::NAT PGAL1Δ:: SpHis5 Sc1068 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 gal4Δ::LEU2 GAL80-2GFP-NAT

Sc1082 MATa ade1 ile leu2-64xLacO-LEU2 This study ura3-52 trp1-HIII his3-Δ1 MEL1 GAL80-2GFP-NAT Sc1084 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-delta1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 GAL80-2GFP-NAT GAL3::LacOx64- LEU2 GAL3:: UASGAL1-10-Kan Sc1122 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15 leu2::LacOx64 LEU2

Sc1123 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15 GAL1::LacOx64 LEU2

continued…

144

…Table 3.9 continued Sc1124 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15 leu2::TetOx512 LEU2

Sc1125 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15 GAL1::TetOx512 LEU2

Sc1130 MATα leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15 leu2::LacOx64 LEU2 Sc1134 MATα leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15 GAL1::TetOx512 LEU2

Sc1153 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 gal80-ΔBglII gal4Δ::LEU2 SEC63- mCherry-KAN Sc1157 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 gal80-ΔBglII GAL4-2GFP-NAT Sc1160 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal4- 2mYFP::NAT GAL1::LacOx64::LEU2

Sc1161 MATa ade1 ile leu2-3, 112 ura3-52 This study trp1-HIII his3-Δ1 MEL1 LYS2::GAL1UAS-GAL1TATA-HIS3 gal80-ΔBglII Gal4-2mYFP::NAT Sc1163 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal4-2GFP::NAT GAL1::LacOx64::LEU2

Continued…

145

…Table 3.9 continued Sc1165 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal4-2GFP::NAT GAL1::LacOx64::LEU2 GAL80- tdTomato-KAN

Sc1198 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3- 1 LacOx64::LEU2:: PGAL1-GST- 24xPP7bs::TRP1 GAL80-2mYFP Sc1205 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal4-2GFP::NAT GAL1::LacOx64::LEU2 GAL80- tdTomato-KAN PGAL1-10-7Δ::SpHIS5

Sc1206 MATa ade1 ile leu2-3,112 ura3-52 This study trp1-HIII his3-Δ1 Gal4-2GFP::NAT GAL1::LacOx64::LEU2 PGAL1-10- 7Δ::KAN JW2687 MATa CSE4-linker-mYFP-kanMX6 Coffman et al. ade2-1 ura3-1 leu2-3, 112 trp1 2011. Gift from his3-11 Wu Lab.

146

Table 3.10: Plasmids used in this study

Plasmid Relevant Genotype Reference/Source

pOE33 CEN6 ARS1 URA3 PGAL1-2GFP Egriboz et al. 2011 pFJ35 CEN6 ARS1 TRP1 PADH2-H2B1- Jiang et al. 2009 mCherry AmpR pME8 CEN6 ARS1 TRP1 PADH2-LacI- Jiang et al. 2009 mCherry AmpR pSG45 CEN6 ARS1 URA3 PSTE50-GAL80- This study 2mYFP AmpR pSG46 CEN6 ARS1 URA3 PSTE50- This study Gal80N230R-2mYFP AmpR pEB1 CEN6 ARS1 TRP1 PGAL4-GAL4DBD- This study Gal80 AmpR pEB2 CEN6 ARS1 TRP1 PGAL4-GAL4DBD- This study Gal80-2mYFP AmpR pSG47 CEN6 ARS1TRP1 PGAL4- This study GAL4DBDL32P-Gal80-2mYFP AmpR pSG48 CEN6 ARS1 TRP1 PGAL4-SV40NLS- This study Gal80-2mYFP AmpR pFJ110N CEN6 ARS1 URA3 PGAL4-Gal4-2GFP- This study NAT AmpR pFJ26N CEN6 ARS1 URA3 PGAL4-Gal4- Jiang et al. 2009 2mYFP-NAT AmpR pSG49 CEN6 ARS1 URA3 PMET25-PP7-2CFP This study AmpR pOE177 YIplac128 LEU2 LacOx64-ChrII- This study 282311-285111 AmpR pFJ58X64 YIplac128 LEU2 LacOx64 AmpR Jiang et al. 2009 pAFS59-TO YIplac128 LEU2 TetOx512 AmpR Bystricky et al. 2004; Gift from Susan Gasser Lab R pGVH29 YIp ADE2 PURA3-TETR-GFP Amp Bystricky et al. 2004; Gift from Susan Gasser Lab pSG50 CEN6 ARS1 TRP1 PURA3-TETR-GFP This study AmpR pSG51 CEN6 ARS1 URA3 PADH2-LacI- This study mCherry AmpR continued… 147 …Table 3.10 continued pDZ276 CEN6 ARS1 URA3 PMET25 PP7- Larson et al. 2yeGFP AmpR 2011; Gift from Singer Lab pOE232 pRS404 –TRP1 PGAL1-GST-24xPP7BS This study AmpR

pRS414 CEN6 ARS1 TRP1 AmpR New England Biolabs pRS416 CEN6 ARS1 URA3 AmpR New England Biolabs R pBM292 CEN6 ARS1 TRP1 PGAL4-GAL4 Amp Johnston and Dover 1986; Gift from Mark Johnston Lab pFJ152 CEN ARS HIS3 PADH2-Nup49- This study mCherry pOE33 CEN ARS1 URA3 PGAL1-2GFP Egriboz et al. 2011

Plasmids were constructed using standard cloning and PCR techniques.

148

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